Herpes simplex viruses and methods of viral replication

ABSTRACT

A herpes simplex virus is disclosed in which the herpes simplex virus genome comprises a nucleic acid sequence encoding an ING4 polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a 35 USC

371 of PCT application Ser. No. PCT/GB2008/000527, filed Feb. 15, 2008,currently pending, entitled “Herpes Simplex Viruses and Methods of ViralReplication,” which claims priority to Great Britain Patent ApplicationNo. 0703066.1, filed Feb. 16, 2007, entitled “Herpes Simplex Viruses andMethods of Viral Replication,” which are each incorporated herein intheir entirety by reference.

FIELD OF THE INVENTION

The present invention relates to Herpes Simplex Viruses, methods for thereplication of Herpes Simplex Virus and the use of Herpes Simplex Virusin the treatment of disease.

Incorporated by reference herein in its entirety is the Sequence Listingco-submitted with the instant application, entitled “6524532.txt”,created Aug. 5, 2009, size of 7 kilobytes.

BACKGROUND

Inhibitor of New Growth 4 (ING 4) is a member of the Inhibitor of NewGrowth family of candidate tumour suppressor proteins of which 6 havebeen reported in humans (Garkavstev et al 1996). Loss of the ING4 genehas been reported in head and neck squamous cell carcinoma (Gunduz et al2005) and in glioma (Garkavtsev et al 2004) and, although a number ofdifferent functions have been described for this protein, its precisemode of action is yet to be elucidated.

The ING gene products inhibit cell proliferation (Russell et al 2006,Campos et al., 2004, Shi and Gozani 2005) and their overexpression isassociated with increased apoptosis (Nagashima et al., (2001). ING4inhibition of cell proliferation is probably mediated by its binding top53 and acetyltransferase p300 thus facilitating acetylation andactivation of p53 (Shiseki et al. 2003). ING4 has been reported as acomponent of the HBO1 HAT complex required for normal cell cycleprogression and the majority of histone H4 acetylation (Doyon et al,2006) suggesting a role in chromatin remodelling and transcriptionalregulation. The extensive loss of histone H4 acetylation during humancancer development strongly suggests a tumour suppressor role for ING4.ING4 has been shown to interact with the RelA subunit of NF-κB resultingin suppressed expression of angiogenesis-related genes such as IL-6,IL-8 and Cox-2 (Garkavtsev et al 2004) and its loss in glioma isassociated with more aggressive tumour growth and vascularisation. Lossof tumour suppressor genes is a common feature of cancer progression.ING4 suppression in multiple myeloma cells in vitro resulted inincreased expression of the pro-angiogenic IL-8 and osteopontin probablyvia increased activity of hypoxia inducible factor-1 (HIF-1), involvedin up-regulating angiogenesis genes during hypoxia, and, in multiplemyeloma patients, decreased levels of ING4 were associated with bothhigh IL-8 production and microvascular density (Colla et al., 2007).ING4 repression of the HIF transcription factor, probably mediated viaan interaction with HIF prolyl hydroxylase (HPH)-2, also involved inregulating angiogenesis genes, has also been reported (Ozer et al.,2005, Colla et al 2007).

Herpes Simplex Virus

The herpes simplex virus (HSV) genome comprises two covalently linkedsegments, designated long (L) and short (S). Each segment contains aunique sequence flanked by a pair of inverted terminal repeat sequences.The long repeat (RL or R_(L)) and the short repeat (RS or R_(S)) aredistinct.

The HSV ICP34.5 (also γ34.5) gene, which has been extensively studied,has been sequenced in HSV-1 strains F and syn17+ and in HSV-2 strainHG52. One copy of the ICP34.5 gene is located within each of the RLrepeat regions. Mutants inactivating both copies of the ICP34.5 gene(i.e. null mutants), e.g. HSV-1 strain 17 mutant 1716 (HSV 1716) or themutants R3616 or R4009 in strain F, are known to lack neurovirulence,i.e. be avirulent (non-neurovirulent), and have utility as both genedelivery vectors or in the treatment of tumours by oncolysis. HSV-1strain 17 mutant 1716 has a 759 bp deletion in each copy of the ICP34.5gene located within the BamHI s restriction fragment of each RL repeat.

ICP34.5 null mutants such as HSV1716 are, in effect, first-generationoncolytic viruses. Most tumours exhibit individual characteristics andthe ability of a broad spectrum first generation oncolytic virus toreplicate in or provide an effective treatment for all tumour types isnot guaranteed.

HSV 1716 is an oncolytic, non-neurovirulent HSV and is described in EP0571410 and WO 92/13943. HSV 1716 has been deposited on 28 Jan. 1992 atthe European Collection of Animal Cell Cultures, Vaccine Research andProduction Laboratories, Public Health Laboratory Services, Porton Down,Salisbury, Wiltshire, SP4 0JG, United Kingdom under accession numberV92012803 in accordance with the provisions of the Budapest Treaty onthe International Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure (herein referred to as the ‘BudapestTreaty’).

SUMMARY OF THE INVENTION

It has now been found that herpes simplex virus comprising nucleic acidencoding an ING4 polypeptide has an enhanced ability to retard tumourgrowth. In particular, the inventors created a variant herpes simplexvirus comprising nucleic acid encoding ING4 polypeptide and investigatedthe effect of administering this variant to mice with tumour implants.They found that the survival time of mice receiving the herpes simplexvirus variant was significantly improved compared to control, and inaddition, the mice receiving the variant had significantly smalleraverage tumour volumes. These results demonstrate that the ING4polypeptide enhances herpes simplex virus-mediated oncolysis.

One possible explanation for these observations is that the ING4polypeptide inhibits angiogenesis, thereby reducing the rate at whichthe tumour can grow. Thus, this mechanism may synergistically interactwith the oncolytic ability of herpes simplex virus to provide animproved tumour cell-killing ability.

However, whilst conducting further investigations the inventorsunexpectedly found that tumour cells infected with the herpes simplexvirus variant comprising nucleic acid encoding ING4 had a significantlyhigher output of progeny virions both in vivo and in vitro compared tocontrol. Further experiments showed that cells constitutively expressingan ING4 polypeptide and infected with wild-type herpes simplex virusalso had a higher output of progeny virions compared to control. Thisindicates that ING4 expression confers a growth advantage on herpessimplex virus.

These results provide the basis for a new approach for improvingefficacy of treatments involving administration of herpes simplex virus.In particular, these results have the potential to lead to much needednew and improved treatments for cancer and other conditions.

HSV1716ING4 has been deposited in the name of Crusade LaboratoriesLimited having an address at PO Box 1716, Glasgow, G51 4WF, UnitedKingdom, at the European Collection of Cell Cultures (ECACC), HealthProtection Agency, Porton Down, Salisbury, Wiltshire, SP4 0JG, UnitedKingdom in accordance with the provisions of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurposes of Patent Procedure (herein referred to as the ‘BudapestTreaty’).

In a broad aspect, the present invention relates to an herpes simplexvirus wherein the herpes simplex virus genome comprises nucleic acidencoding an anti-angiogenic polypeptide or protein. In other broadaspects, the invention relates to an herpes simplex virus wherein theherpes simplex virus genome comprises nucleic acid encoding apolypeptide that enhances replication efficiency of the herpes simplexvirus. The anti-angiogenic polypeptide may be a polypeptide thatenhances HSV replication efficiency.

The anti-angiogenic polypeptide or protein, or polypeptide that enhancesHSV replication efficiency, is preferably heterologous to the HSV, i.e.not being normally encoded or expressed by the corresponding wild typeHSV. More preferably, the anti-angiogenic polypeptide or protein, orpolypeptide that enhances HSV replication efficiency, is a mammalianpolypeptide or protein. Still more preferably it is a candidate tumorsuppressor gene. The HSV is preferably capable of expressing theanti-angiogenic polypeptide or polypeptide that enhances HSV replicationefficiency. The anti-angiogenic polypeptide or polypeptide that enhancesHSV replication efficiency is preferably an ING4 polypeptide.

More particularly, the present invention concerns HSV capable ofexpressing ING4 and the use of ING4 in improving the replicationefficiency of HSV.

Accordingly, in one aspect, the present invention relates to a herpessimplex virus, wherein the herpes simplex virus genome comprises anucleic acid sequence encoding an anti-angiogenic polypeptide. In apreferred embodiment the anti-angiogenic polypeptide is ING4. The herpessimplex virus may also be non-neurovirulent.

In a further aspect, the present invention relates to a herpes simplexvirus wherein the herpes simplex virus genome comprises a nucleic acidsequence encoding an ING4 polypeptide. The herpes simplex virus may benon-neurovirulent.

The HSV may be an oncolytic HSV. HSV, e.g. oncolytic HSV, that may beused in the invention include HSV in which one or both of the γ34.5(also called ICP34.5) genes are modified (e.g. by mutation which may bea deletion, insertion, addition or substitution) such that therespective gene is incapable of expressing, e.g. encoding, a functionalICP34.5 protein. Preferably, in HSV according to the invention bothcopies of the γ34.5 gene are modified such that the modified HSV is notcapable of expressing, e.g. producing, a functional ICP34.5 protein. Theviruses, e.g. oncolytic viruses, are preferably non-neurovirulent.

In certain arrangements the herpes simplex virus may be a gene specificnull mutant, such as an ICP34.5 null mutant. Where all copies of theICP34.5 gene present in the herpes simplex virus genome (two copies arenormally present) are disrupted such that the herpes simplex virus isincapable of producing a functional ICP34.5 gene product, the virus isconsidered to be an ICP34.5 null mutant. In other arrangements theherpes simplex virus may lack at least one expressible ICP34.5 gene. Inanother arrangement the herpes simplex virus may lack only oneexpressible ICP34.5 gene. In other arrangements the herpes simplex virusmay lack both expressible ICP34.5 genes. In still other arrangementseach ICP34.5 gene present in the herpes simplex virus may not beexpressible. Lack of an expressible ICP34.5 gene means, for example,that expression of the ICP34.5 gene does not result in a functionalICP34.5 gene product.

The genome of the HSV according to the invention is further modified tocontain nucleic acid encoding at least one copy of an anti-angiogenicpolypeptide such that the polypeptide can be expressed from the nucleicacid. The nucleic acid encoding the polypeptide may be located in atleast one RL1 locus of the herpes simplex virus. For example, thenucleic acid may be located in or overlap at least one of the ICP34.5protein coding sequences of the herpes simplex virus genome. Thisprovides a convenient way of inactivating the ICP34.5 gene, therebyproviding non-neurovirulence.

The anti-angiogenic polypeptide or polypeptide that enhances replicationefficiency may be a selected ING4 polypeptide. As such, in a preferredembodiment the modified HSV is an expression vector capable ofexpressing the anti-angiogenic polypeptide or protein, or HSVpolypeptide that enhances replication efficiency (e.g. ING4) uponinfection of cells, preferably mammalian cells. In another preferredembodiment, the HSV is an oncolytic expression vector.

In order to effect expression of the anti-angiogenic polypeptide orpolypeptide that enhances replication efficiency (e.g. ING4) the nucleicacid encoding the polypeptide is preferably operably linked to aregulatory sequence, e.g. a promoter, capable of effecting transcriptionof the nucleic acid encoding the polypeptide. A regulatory sequence(e.g. promoter) that is operably linked to a nucleotide sequence may belocated adjacent to that sequence or in close proximity such that theregulatory sequence can effect and/or control expression of a product ofthe nucleotide sequence. The encoded product of the nucleotide sequencemay therefore be expressible from that regulatory sequence.

In this specification the term “operably linked” may include thesituation where a selected nucleotide sequence and regulatory nucleotidesequence are covalently linked in such a way as to place the expressionof a nucleotide sequence under the influence or control of theregulatory sequence. Thus a regulatory sequence is operably linked to aselected nucleotide sequence if the regulatory sequence is capable ofeffecting transcription of a nucleotide sequence which forms part or allof the selected nucleotide sequence. Where appropriate, the resultingtranscript may then be translated into a desired protein or polypeptide.

Nucleic acid vectors useful for generating herpes simplex viruses of thepresent invention are described, for example, on page 41 line 19 to page55 line 30 of WO 2005/049845. This is incorporated herein by reference.One such vector provided by the inventors is plasmid RL1.dIRES-GFPdeposited in the name of Crusade Laboratories Limited at the EuropeanCollection of Cell Cultures (ECACC), Health Protection Agency, PortonDown, Salisbury, Wiltshire, SP4 0JG, United Kingdom on 3 Sep. 2003 underaccession number 03090303 in accordance with the provisions of theBudapest Treaty.

The nucleic acid sequence encoding the ING4 polypeptide may form part ofa nucleic acid cassette which is inserted in the genome of a selectedherpes simplex virus by homologous recombination. Whether part of acassette or not, the site of insertion may be in any genomic locationselected. One preferred insertion site is in one or both of the longrepeat regions (R_(L)), and one copy of the cassette is preferablyinserted in each copy of the long repeat (R_(L)). More preferably theinsertion site is in at least one (preferably both) RL1 locus and mostpreferably it is inserted in at least one (preferably both) of theICP34.5 protein coding sequences of the HSV genomic DNA. It is preferredthat the insertion occurs in identical or substantially similarpositions in each of the two repeat regions, RL1 loci or ICP34.5 proteincoding sequences.

Insertion may be such as to produce a modified virus which is anon-neurovirulent mutant capable of expressing the encoded ING4polypeptide upon transfection into mammalian, more preferably human,cells in vivo and in vitro. The non-neurovirulent mutant may be anICP34.5 null mutant.

The nucleic acid cassette may be of any size, e.g. up to 5, 10, 15, 20,25, 30, 35, 40, 45 or 50 kbp in length.

HSV according to the invention are provided for use in medical treatmentof disease, more particularly in the treatment of a cancerous condition.A method of treatment of a cancerous condition is provided comprisingthe step of administering an HSV according to the invention to anindividual in need of such treatment, e.g. in a therapeuticallyeffective amount. HSV according to the invention are also provided foruse in the manufacture of a medicament for the treatment of a cancerouscondition. HSV according to the invention are also provided for use intreatment of a tumour, e.g. oncolytic treatment of a tumour.

A medicament, pharmaceutical composition or vaccine comprising an herpessimplex virus according to the present invention is also provided whichmay comprise an HSV according to the present invention together with apharmaceutically acceptable carrier, adjuvant or diluent.

The herpes simplex virus of the invention may be derived from any HSVincluding any laboratory strain or clinical isolate (non-laboratorystrain) of HSV. Preferably the HSV is a mutant of HSV-1 or HSV-2.Alternatively the HSV may be an intertypic recombinant of HSV-1 andHSV-2. The mutant may be of one of laboratory strains HSV-1 strain 17,HSV-1 strain F or HSV-2 strain HG52. The mutant may be of thenon-laboratory strain JS-1. Preferably the mutant is a mutant of HSV-1strain 17. The herpes simplex virus may be a further mutant of one ofHSV-1 strain 17 mutant 1716, HSV-1 strain F mutant R3616, HSV-1 strain Fmutant G207, HSV-1 mutant NV1020. Preferably the herpes simplex virus isa further mutant of one of HSV-1 strain 17 mutant 1716.

Herpes simplex viruses of the invention may be used in ‘gene delivery’methods in vitro or in vivo. Non-neurovirulent herpes simplex viruses ofthe invention are expression vectors and may be used to infect selectedcells or tissues in order to express the anti-angiogenic polypeptide orprotein, or polypeptide that enhances HSV replication efficiency (e.g.ING4) encoded by the herpes simplex virus genome.

In one arrangement, cells may be taken from a patient, a donor or fromany other source, infected with a herpes simplex virus of the invention,optionally screened for expression and/or function of the encoded ING4,and optionally returned/introduced to a patient's body, e.g. byinjection.

Delivery of herpes simplex viruses of the invention to the selectedcells may be performed using naked virus or by encapsulation of thevirus in a carrier, e.g. nanoparticles, liposomes or other vesicles.

In vitro cultured cells, preferably human or mammalian cells,transformed with viruses of the present invention and preferably cellsexpressing the ING4 polypeptide as well as methods of transforming suchcells in vitro with said viruses form further aspects of the presentinvention.

The HSV genome may contain additional mutations and/or heterologousnucleotide sequences. Additional mutations may include disablingmutations, which may affect the virulence of the virus or its ability toreplicate. For example, mutations may be made in any one or more ofICP6, ICP0, ICP4, ICP27. Preferably, a mutation in one of these genes(optionally in both copies of the gene where appropriate) leads to aninability (or reduction of the ability) of the HSV to express thecorresponding functional polypeptide. By way of example, the additionalmutation of the HSV genome may be accomplished by addition, deletion,insertion or substitution of nucleotides.

The cancerous condition may be any unwanted cell proliferation (or anydisease manifesting itself by unwanted cell proliferation), neoplasm ortumour or increased risk of or predisposition to the unwanted cellproliferation, neoplasm or tumour. The cancerous condition may be acancer and may be a benign or malignant cancer and may be primary orsecondary (metastatic). A neoplasm or tumour may be any abnormal growthor proliferation of cells and may be located in any tissue. Examples oftissues include the colon, pancreas, lung, breast, uterus, stomach,kidney, testis, central nervous system (including the brain), peripheralnervous system, skin, blood or lymph.

Cancer/tumour types which may be treated may be primary or secondary(metastatic) tumours. Tumours to be treated may be nervous systemtumours originating in the central or peripheral nervous system, e.g.glioma, medulloblastoma, meningioma, neurofibroma, ependymoma,Schwannoma, neurofibrosarcoma, astrocytoma and oligodendroglioma, or maybe non-nervous system tumours originating in non-nervous system tissuee.g. melanoma, mesothelioma, lymphoma, hepatoma, epidermoid carcinoma,prostate carcinoma, breast cancer cells, lung cancer cells or coloncancer cells. HSV mutants of the present invention may be used to treatmetastatic tumours of the central or peripheral nervous system whichoriginated in a non-nervous system tissue. For example, cancerousconditions that may be treated by the invention include squamous cellcarcinomas, ovarian tumours/carcinomas, hepatocellular carcinomas, andbreast adenocarcinomas.

In this specification, non-neurovirulence is defined by the ability tointroduce a high titre of virus (approx 10⁶ plaque forming units (pfu))to an animal or patient without causing a lethal encephalitis such thatthe LD₅₀ in animals, e.g. mice, or human patients is in the approximaterange of ≧10⁶ pfu.

The patient to be treated may be any animal or human. The patient may bea non-human mammal, but is more preferably a human patient. The patientmay be male or female.

The invention also provides a method of lysing or killing tumour cellsin vitro or in vivo comprising the step of administering to the cells anherpes simplex virus of the invention.

The invention also provides a method of expressing in vitro or in vivoan ING4 polypeptide, said method comprising the step of infecting atleast one cell or tissue of interest with a herpes simplex virus of theinvention.

In a further aspect, the invention relates to an in vivo or in vitromethod of increasing replication efficiency of a herpes simplex virus ina cell infected with herpes simplex virus, comprising the step ofcausing the cell to express a nucleic acid sequence encoding an ING4polypeptide.

The increase in replication may be an increase in the amount of virionsproduced by the cell, e.g. an increase in HSV yield. The increase in HSVreplication efficiency may be relative to control, e.g. relative to acell that expresses ING4 at wild-type levels. The step of causing, e.g.stimulating, the cell to express a nucleic acid sequence encoding anING4 polypeptide may comprise causing and/or effecting an increase inthe concentration of ING4 polypeptide in the cell, e.g. relative tocontrol. For example, the method may comprise increasing expression ofnucleic acid encoding ING4 polypeptide. This may be achieved byintroducing into the cell a heterologous construct comprising thenucleic acid sequence encoding an ING4 polypeptide, e.g. the termheterologous meaning that the construct is not normally present in thewild-type cell. The nucleic acid sequence encoding an ING4 polypeptidemay be operably linked to a regulatory nucleotide sequence, wherein saidregulatory nucleotide sequence has a role in controlling transcriptionof the ING4 polypeptide. Expression of the nucleic acid encoding ING4may be constitutive.

The presence of ING4 in a cell (at levels above the endogenous level ofING4 normally present in the cell type) infected with HSV can lead to anincrease in replication efficiency of the HSV. This may result in agreater yield of HSV virions from the infected cell(s). Replicationefficiency can be measured by comparing the number of virions producedby cells infected with HSV and in which excess ING4 (i.e. ING4additional to that normally present because of endogenous expression) ispresent with control cells infected with the same HSV but in whichexcess ING4 is not present, i.e. control cells are infected with thesame HSV but do not produce ING4 polypeptide above the normal endogenouslevel. One may optionally determine a value for the number of HSVvirions produced per pfu of HSV infected for cells in which ING4 is andis not present. An increase in replication efficiency is present wherecells in which excess ING4 is present show an increase, preferably astatistically significant increase, in the amount of HSV virionsproduced compared to the control. The increase in replication efficiencymay lead to production level of virions that is 1.1, 1.2, 1.3, 1.4, 1.5,2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200,300, 400, 500, 600, 700, 800, 900 or 1000 (or more) times that of thecontrol.

The cell may be any cell that is capable of being infected with HSV, butis preferably a tumour cell, e.g. tumour cells capable of being lysed byHSV, e.g. squamous cell carcinoma, ovarian tumour, human breastadenocarcinoma, ovarian carcinoma and hepatocellular carcinoma.

In a further aspect the invention provides a cell, in vivo or in vitro,infected with a herpes simplex virus, which cell comprises aheterologous construct comprising nucleic acid sequence encoding an ING4polypeptide. Expression of the nucleic acid encoding ING4 may beconstitutive.

In a further aspect the invention provides a nucleic acid encoding anING4 polypeptide for use in a therapeutic method, which method comprisesadministering a non-neurovirulent herpes simplex virus to a patient inneed of treatment. In a further aspect, the invention provides anon-neurovirulent herpes simplex virus for use in a therapeutic method,which method comprises administering a nucleic acid encoding ING4 topatient in need of treatment.

In a further aspect the invention provides use of a nucleic acidencoding an ING4 polypeptide in the manufacture of a medicament for atherapeutic method, which method comprises administering anon-neurovirulent herpes simplex virus to patient in need of treatment.In a further aspect the invention provides use of a non-neurovirulentherpes simplex virus in the manufacture of a medicament for atherapeutic method, which method comprises administering a nucleic acidencoding ING4 to patient in need of treatment.

In a further aspect the invention provides a method of treating apatient comprising administering to the patient a non-neurovirulentherpes simplex virus and a nucleic acid encoding an ING4 polypeptide.

The patient in need of treatment may be a patient with a cancerouscondition. The treatment may be treatment of cancer and/or tumour.

Nucleic acid encoding an ING4 polypeptide for use in methods oftreatment may be provided in a vector. The vector may be a gene therapyvector, e.g. it may enter cells of the patient upon administration. Forexample, the vector may be a virus vector, e.g. it may be anon-oncolytic HSV.

In further aspect the invention provides a composition comprising aherpes simplex virus and a nucleic acid encoding an ING4 polypeptideand/or an ING4 polypeptide. The composition may, for example, be apharmaceutical composition comprising a non-neurovirulent herpes simplexvirus and a nucleic acid encoding an ING4 polypeptide.

The inventors have shown that introduction of excess ING4 polypeptide toa cell (i.e. additional ING4 beyond that normally expressed by the cell)leads to increased levels of expression of wild type and mutant HSV.Accordingly, the present invention provides a method of replicating HSVin vitro or in vivo comprising (i) providing a cell or cells) havingexcess ING 4 and (ii) contacting the cell(s) with HSV capable ofinfecting the cell(s). For in vitro production of HSV via replication ininfected cells this approach may lead to significant improvements inreplication of HSV and enable higher viral titres to be obtained. Themethod may comprise the step of causing the cell(s) to express a nucleicacid sequence encoding an ING4 polypeptide. This may comprise the stepof introducing into the cell(s) a heterologous construct comprising anucleic acid sequence encoding an ING4 polypeptide.

Excess ING4 may be introduced to cells by causing the cell to expressING4 from a vector transfected into the cell, e.g. expression ofrecombinant ING4 from an expression vector. The vector may be an HSVsuch as HSV1716ING4 but may also be any suitable expression vector, e.g.plasmid vector. Additionally or alternatively the cell may be caused tooverexpress endogenous ING4, e.g. through regulation or enhancement ofthe corresponding promoter. Additionally or alternatively the cell maybe contacted with ING4 polypeptide, which for example may be obtained asrecombinant ING4, e.g. through fermentation of bacteria transfected withan expression vector encoding ING4.

Accordingly, a further aspect of the present invention provides a methodof replicating herpes simplex virus in cells infected with herpessimplex virus comprising contacting the cell with ING4 polypeptide. In afurther aspect the invention provides a method of replicating herpessimplex virus in cells infected with herpes simplex virus comprisingcontacting the cell with a nucleic acid vector encoding ING4 and causingthe cell to express ING4.

In accordance with the above, in yet a further aspect a method isprovided for the in vitro replication of HSV comprising the steps of (i)providing a cell or cells with excess ING4, (ii) infecting the cell(s)with HSV, (iii) culturing the cell(s) in vitro, and (iv) harvestingviral progeny from the cell(s).

The HSV may be any HSV as described herein.

The cells may be any cells capable of infection by HSV and in which HSVcan replicate. They may be in vitro cultured cells. They may be human ornon-human cells. For example they may be from rabbit, guinea pig, rat,mouse or other rodent (including cells from any animal in the orderRodentia), cat, dog, pig, sheep, goat, cattle, horse, non-human primateor other non-human vertebrate organism; and/or non-human mammaliancells; and/or human cells.

In a further aspect of the invention, the invention providesHSV1716ING4, which has been deposited with the ECACC (EuropeonCollection of Cell Cultures, address Health Protection Agency, PortonDown, Salisbury, Wiltshire SP4 0JG, United Kingdom, in accordance withthe Budapest Treaty on Feb. 15, 2008 under accession no. 08021501.

ING4

Reference to ING4 in this specification includes reference to the ING4polypeptide having the amino acid sequence described under Accession no.NM_(—)016162 GI:38201669 in the NCBI database at ncbi.nlm.nih.gov.Reference to ING4 also includes reference to isoforms, homologues andderivatives of ING4. Derivatives may comprise natural variations orpolymorphisms which may exist between individuals or between members ofa family. There are many homologues of ING4 polypeptide deposited in theGenbank database. For example a Blast search of the database identifiedaround one hundred homologues. Homologues include, for example, otherhuman ING4 isoforms such as BC007781, Mus musculus ING4 (e.g.NP_(—)579923.1), Cannis familiaris ING4 (XP_(—)534907.2), Rattusnorvegicus (NP_(—)001073356.1), Pan troglodytes ING4(XP_(—)001169091.1), Equus caballus (XP_(—)001496617.1), Gallus gallusING4 (NP_(—)001006241.1), Macaca mulatta (XP_(—)001118270.1),Monodelphis domestica (XP_(—)001365016.1), Bos Taurus(NP_(—)001030466.1), Danio rerio ((NP_(—)001018304.1), Xonpus laevis(NP_(—)001088224.1). The ING4 polypeptide or protein to which theinvention relates is preferably a human ING4.

All such homologues and derivatives are included within the scope of theinvention. Purely as examples, conservative replacements which may befound in such polymorphisms may be between amino acids within thefollowing groups:

-   -   (i) alanine, serine, threonine;    -   (ii) glutamic acid and aspartic acid;    -   (iii) arginine and leucine;    -   (iv) asparagine and glutamine;    -   (v) isoleucine, leucine and valine;    -   (vi) phenylalanine, tyrosine and tryptophan.

In particular, peptides and polypeptides having a sequence identity ofat least 70%, more preferably one of 75%, 80%, 85%, 90%, 95%, 96%, 97%,98%, 99% and 100% with the amino acid sequence of ING4 set out underAccession no. NM_(—)016162 GI:38201669 are included as ING4 polypeptidesfor the purposes of this specification. Nucleic acid sequences encodingany one of these ING4 polypeptides may be used for the purposes of ING4expression in accordance with this invention.

Nucleic acid encoding an ING4 polypeptide may be selected by its abilityto hybridise to the nucleic acid of SEQ ID NO: 4 or SEQ ID NO: 5 underhigh stringency conditions.

The ING4 polypeptide may be a polypeptide whose presence in a cellinfected with herpes simplex virus leads to an increase in replicationefficiency of the herpes simplex virus in said cell. In particular, theING4 polypeptide may be a polypeptide whose presence in a cell infectedwith herpes simplex virus strain 17 or 1716 leads to an increase inreplication efficiency of herpes simplex virus strain 17 or 1716 in saidcell.

For example, an increase in expression of the nucleic acid sequenceencoding the ING4 polypeptide in a cell infected with a herpes simplexvirus leads to an increase in replication efficiency of the herpessimplex virus. As described above, an increase in replicationefficiency, e.g. a greater replication efficiency, means, for example,that the cell produces a greater number of progeny virions, e.g. theyield of HSV particles from the cell is greater. The increase inreplication efficiency may be relative to control, e.g. relative to acell which expresses endogenous levels, or does not express, ING4. Forexample, where the HSV genome comprises nucleic acid encoding ING4polypeptide, the increase in HSV replication efficiency may be relativeto the same cell infected with the corresponding HSV that does notcomprise nucleic acid encoding ING4 polypeptide.

The HSV may be strain 17 or strain 1716. Preferably the increase inreplication efficiency is statistically significant, e.g. p<0.05, e.g.as determined using the Student's t-test or analysis of variance(ANOVA).

The increase in expression may be increase in expression in a cell lineselected from the group consisting of: BHK, A431 human squamous cellcarcinoma, CP70 human ovarian tumour, MDA-MB-468 human breastadenocarcinoma, Ovcar 3 human ovarian carcinoma and HuH7 humanhepatocellular carcinoma. The experiments in Example 2 set out asuitable procedure to compare replication efficiency. For example,tumour cells may be infected with 1000 pfu for 72 hours.

The ING4 nucleotide sequence may encode a full length transcript orpolypeptide (i.e. comprise the complete ING4 protein coding sequence).Alternatively, provided the polypeptide product retains ING4 activity,e.g. increased HSV replication efficiency, the ING4 nucleotide sequencemay comprise one or more fragments of the full length sequencerespectively coding for a fragment of the full length transcript or atruncated polypeptide.

A fragment may comprise a nucleotide sequence encoding at least 10% ofthe corresponding full length sequence, more preferably the fragmentcomprises at least 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96, 97, 98 or99% of the corresponding full length sequence. Preferably, the fragmentcomprises at least, i.e. has a minimum length of, 20 nucleotides, morepreferably at least 30, 40, 50, 100, 150, 200, 300, 400, 500, 600, 700,800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or2000 nucleotides. The fragment may have a maximum length, i.e. be nolonger than, 20 nucleotides, more preferably no longer than 30, 40, 50,100, 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200,1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 nucleotides. Thefragment length may be anywhere between said minimum and maximum length.

In one preferred arrangement, the herpes simplex virus is HSV1716ING4deposited in the name of Crusade Laboratories Limited having an addressat PO Box 1716, Glasgow, G51 4WF, United Kingdom, at the EuropeanCollection of Cell Cultures (ECACC), Health Protection Agency, PortonDown, Salisbury, Wiltshire, SP4 0JG, United Kingdom in accordance withthe provisions of the Budapest Treaty.

The amino acid and nucleotide sequences for human ING4 as set out underAccession no. NM_(—)016162.2 GI:38201669 of the NCBI database arereproduced below as SEQ ID No. 3 and SEQ ID No.4.

Amino acid sequence: [SEQ ID No. 33]MAAGMYLEHYLDSIENLPFELQRNFQLMRDLDQRTEDLKAEIDKLATEYMSSARSLSSEEKLALLKQIQEAYGKCKEFGDDKVQLAMQTYEMVDKHIRRLDTDLARFEADLKEKQIESSDYDSSSSKGKKSRTQKEKKAARARSKGKNSDEEAPKTAQKKLKLVRTSPEYGMPSVTFGSVHPSDVLDMPVDPNEPTYCLCHQVSYGEMIGCDNPDCSIEWFHFACVGLTTKPRGKWFCPRCSQERKKK Nuclectide sequence: [SEQ ID No. 4] 1ccggggcgga tcggaagttg ctttgttttg cttcgagatg gctgcgggga tgtatttgga 61acattatctg gacagtattg aaaaccttcc ctttgaatta cagagaaact ttcagctcat 121gagggaccta gaccaaagaa cagaggacct gaaggctgaa attgacaagt tggccactga 181gtatatgagt agtgcccgca gcctgagctc cgaggaaaaa ttggcccttc tcaaacagat 241ccaggaagcc tatggcaagt gcaaggaatt tggtgacgac aaggtgcagc ttgccatgca 301gacctatgag atggtggaca aacacattcg gcggctggac acagacctgg cccgttttga 361ggctgatctc aaggagaaac agattgagtc aagtgactat gacagctctt ccagcaaagg 421caaaaagagc cggactcaaa aggagaagaa agctgctcgt gctcgttcca aagggaaaaa 481ctcggatgaa gaagccccca agactgccca gaagaagtta aagctcgtgc gcacaagtcc 541tgagtatggg atgccctcag tgacctttgg cagtgtccac ccctctgatg tgttggatat 601gcctgtggat cccaacgaac ccacctattg cctttgtcac caggtctcct atggagagat 661gattggctgt gacaaccctg attgttccat tgagtggttc cattttgcct gtgtggggct 721gacaaccaag cctcggggga aatggttttg cccacgctgc tcccaagaac ggaagaagaa 781atagataagg gccttggatt ccaacacagt ttcttccaca tcccctgact tgggctagtg 841ggcagaggaa tgcctgtgct ggggccaggg gttcagggag gagtggatgg cacagtgctg 901tcatcccttc tcctcccctc tccccactcc cggtgctgag gctgcatcag accctggtag 961ggaggggtgc cgcagccact aacggtatgt gctctccttc agccctctcc cttcggaggg 1021acgtggtctt gcccactgtc cttttgcctc catgctgagg tcggtgctgt atttcagagg 1081gagggtcctt ttcattctcc ttgctttgta tttaaggact ggggcatagc atgggggcag 1141tcccccagac ctcttcattc cccctcctgt ggtgagggct aggtgtgatc aacacttttc 1201ttctccattc ccttcctgct tttttcatgg tgggggatcc accaggtcat ctaggctctg 1261gccctagttg aaggggcacc ccttcctctg tgccaagagg attcatcctg ggagaggggg 1321caaggtggaa tgcagataac tcacatgtaa aaggaacttg ggtaggtaaa taaaagctat 1381acatgttggc ctgctgtgtt tattgtagag acactgtttt agtaaacatg ctgagcattc 1441attttgcgtc ctctgggttg gatgcaatgt gagaggatgg catgccagaa ttaggacacg 1501acatgaaacc agagtggtgc ctctgtccga gaacttgtaa gttctcaact tgggaaagac 1561agaggtgctg gagggtaggc ctcagaccag ggggtctcca aaactttgta aatcatgcat 1621cttttctcca taaaacatct ttcacttaat ttccaataaa tgatgtattt gtgctataca 1681tacgtactgc tatactataa aaaaaaaaaa aaaaaaa

The 747 bp cDNA sequence encoding human ING4 (SEQ ID No.5) is foundbetween positions 38-784 (inclusive) in SEQ ID No.4.

Herpes Simplex Viruses

In this specification a herpes simplex virus (HSV) may be any herpessimplex virus. Suitable HSV include any laboratory strain or clinicalisolate (non-laboratory strain) of HSV. Preferably the HSV is an HSV-1or HSV-2. Alternatively the HSV may be an intertypic recombinant ofHSV-1 and HSV-2. The HSV may be one of laboratory strains HSV-1 strain17, HSV-1 strain F or HSV-2 strain HG52. The HSV may be thenon-laboratory strain JS-1. Preferably the HSV is HSV-1 strain 17 or amutant thereof. The HSV may be a further mutant of one of HSV-1 strain17 mutant 1716, HSV-1 strain F mutant R3616, HSV-1 strain F mutant G207,HSV-1 mutant NV1020.

The parent herpes simplex virus, from which a virus of the invention isderived may be of any kind, e.g. HSV-1 or HSV-2. In one preferredarrangement the herpes simplex virus is a variant of HSV-1 strain 17 andmay be obtained by modification of the strain 17 genomic DNA. Suitablemodifications include the insertion of the nucleic acid sequenceencoding the angi-angiogenic polypeptide or polypeptide that enhancesreplication efficiency into the herpes simplex virus genomic DNA. Theinsertion may be performed by homologous recombination of the exogenousnucleic acid sequence into the genome of the selected herpes simplexvirus.

Although the non-neurovirulent phenotype of the herpes simplex virus ofthe invention may be the result of insertion of the nucleic acidsequence in the RL1 locus, herpes simplex viruses according to thepresent invention may be obtained by utilising a non-neurovirulentparent strain, e.g. HSV1716 deposited under the Budapest Treaty at theEuropean Collection of Animal Cell Cultures (ECACC), Health ProtectionAgency, Porton Down, Salisbury, Wiltshire, United Kingdom underaccession number V92012803, and inserting the nucleic acid sequence atanother location of the genome by standard genetic engineeringtechniques, e.g. homologous recombination. In this aspect the locationof the herpes simplex virus genome selected for insertion of the ING4nucleic acid sequence or cassette containing said sequence may be aneutral location.

Herpes simplex viruses of the present invention may be variants of aknown ‘parent’ strain from which the herpes simplex virus of theinvention has been derived. A particularly preferred parent strain isHSV-1 strain 17. Other parent strains may include HSV-1 strain F orHSV-2 strain HG52 or any of the laboratory or non-laboratory strainsdescribed above or any other HSV. A variant comprises an HSV in whichthe genome substantially resembles that of the parent, contains thenucleic acid sequence and may contain a limited number of othermodifications, e.g. one, two, three, four, five or less than ten otherspecific mutations, which may be introduced to disable the pathogenicproperties of the herpes simplex virus, for example a mutation in theribonucleotide reductase (RR) gene, the 65K trans inducing factor (TIF)and/or a small number of mutations resulting from natural variation,which may be incorporated naturally during replication and selection invitro or in vivo. Otherwise the genome of the variant will be that ofthe parent strain.

For example, in some embodiments the herpes simplex virus genome may,have a mutation in the ICP34.5 gene and the ribonucleotide reductasegene, e.g. the ICP34.5 and ribonucleotide reductase genes may notproduce a functional product, but the genome may otherwise substantiallyresemble the genome of HSV-1 strain 17 or F or HSV-2 strain HG52. Inother embodiments the herpes simplex virus genome may have a mutation inthe ICP34.5 gene and ribonucleotide reductase gene, e.g. the ICP34.5 andribonucleotide reductase gene may not produce a functional product, butthe genome may otherwise be the genome of HSV-1 strain 17 or F or HSV-2strain HG52. The term “substantially resembles” may mean that the herpessimplex virus genome is 70, 75, 80, 85, 90, 95, 96, 97, 98, 99, 99.9 or99.99% identical to the genome of HSV-1 strain 17 or F or HSV-2 strainHG52.

The herpes simplex virus of the present invention is preferably a mutantherpes simplex virus. A mutant herpes simplex virus is a non-wild typeherpes simplex virus and may be a recombinant herpes simplex virus.Mutant herpes simplex viruses may comprise a genome containingmodifications relative to the wild type. A modification may include atleast one deletion, insertion, addition or substitution.

As described above, the herpes simplex virus may be a laboratory HSVstrain or a non-laboratory strain. The laboratory strain may be, forexample, HSV-1 strain 17, or F, or HSV-2 strain HG52. Laboratory strainsmay optionally be serially passaged HSV strains, e.g. a strain that hasbeen serially passaged at least 100, 200, 500, 1000, or 10000 times. Anon-laboratory strain may be a clinical isolate, e.g. a recent clinicalisolate. A laboratory strain is, for example, not a recent clinicalisolate.

Sequence Identity

Percentage (%) sequence identity is defined as the percentage of aminoacid residues in a candidate sequence that are identical with residuesin the given listed sequence (referred to by the SEQ ID No.) afteraligning the sequences and introducing gaps if necessary, to achieve themaximum sequence identity, and not considering any conservativesubstitutions as part of the sequence identity. Sequence identity ispreferably calculated over the entire length of the respectivesequences.

Where the aligned sequences are of different length, sequence identityof the shorter comparison sequence may be determined over the entirelength of the longer given sequence or, where the comparison sequence islonger than the given sequence, sequence identity of the comparisonsequence may be determined over the entire length of the shorter givensequence.

For example, where a given sequence comprises 100 amino acids and thecandidate sequence comprises 10 amino acids, the candidate sequence canonly have a maximum identity of 10% to the entire length of the givensequence. This is further illustrated in the following example:

(A) Given seq: XXXXXXXXXXXXXXX (15 amino acids) Comparison seq:XXXXXYYYYYYY (12 amino acids)

The given sequence may, for example, be that encoding ING4 (e.g. SEQ IDNo.3).

% sequence identity=the number of identically matching amino acidresidues after alignment divided by the total number of amino acidresidues in the longer given sequence, i.e. (5 divided by 15)×100=33.3%

Where the comparison sequence is longer than the given sequence,sequence identity may be determined over the entire length of the givensequence. For example:

(B) Given seq: XXXXXXXXXX (10 amino acids) ComparisonXXXXXYYYYYYZZYZZZZZZ (20 amino acids) seq:

Again, the given sequence may, for example, be that encoding ING4 (e.g.SEQ ID No.3).

% sequence identity=number of identical amino acids after alignmentdivided by total number of amino acid residues in the given sequence,i.e. (5 divided by 10)×100=50%.

Alignment for purposes of determining percent amino acid sequenceidentity can be achieved in various ways known to a person of skill inthe art, for instance, using publicly available computer software suchas ClustalW 1.82. T-coffee or Megalign (DNASTAR) software. When usingsuch software, the default parameters, e.g. for gap penalty andextension penalty, are preferably used. The default parameters ofClustalW 1.82 are: Protein Gap Open Penalty=10.0, Protein Gap ExtensionPenalty=0.2, Protein matrix=Gonnet, Protein/DNA ENDGAP=−1, Protein/DNAGAPDIST=4.

Identity of nucleic acid sequences may be determined in a similar mannerinvolving aligning the sequences and introducing gaps if necessary, toachieve the maximum sequence identity, and calculating sequence identityover the entire length of the respective sequences. Where the alignedsequences are of different length, sequence identity may be determinedas described above and illustrated in examples (A) and (B).

Hybridisation Stringency

In accordance with the present invention, nucleic acid sequences may beidentified by using hybridization and washing conditions of appropriatestringency.

Complementary nucleic acid sequences will hybridise to one anotherthrough Watson-Crick binding interactions. Sequences which are not 100%complementary may also hybridise but the strength of the hybridisationusually decreases with the decrease in complementarity. The strength ofhybridisation can therefore be used to distinguish the degree ofcomplementarity of sequences capable of binding to each other.

The “stringency” of a hybridization reaction can be readily determinedby a person skilled in the art.

The stringency of a given reaction may depend upon factors such as probelength, washing temperature, and salt concentration. Higher temperaturesare generally required for proper annealing of long probes, whileshorter probes may be annealed at lower temperatures. The higher thedegree of desired complementarity between the probe and hybridisablesequence, the higher the relative temperature which can be used. As aresult, it follows that higher relative temperatures would tend to makethe reaction conditions more stringent, while lower temperatures lessso.

For example, hybridizations may be performed, according to the method ofSambrook et al., (“Molecular Cloning, A Laboratory Manual, Cold SpringHarbor Laboratory Press, 1989) using a hybridization solutioncomprising: 5×SSC, 5×Denhardt's reagent, 0.5-1.0% SDS, 100 μg/mldenatured, fragmented salmon sperm DNA, 0.05% sodium pyrophosphate andup to 50% formamide. Hybridization is carried out at 37-42° C. for atleast six hours. Following hybridization, filters are washed as follows:(1) 5 minutes at room temperature in 2×SSC and 1% SDS; (2) 15 minutes atroom temperature in 2×SSC and 0.1% SDS; (3) 30 minutes-1 hour at 37° C.in 1×SSC and 1% SDS; (4) 2 hours at 42-65° C. in 1×SSC and 1% SDS,changing the solution every 30 minutes.

One common formula for calculating the stringency conditions required toachieve hybridization between nucleic acid molecules is to calculate themelting temperature T_(m) (Sambrook et al., 1989):T _(m)=81.5° C.+16.6 Log [Na+]+0.41(% G+C)−0.63(% formamide)−600/nwhere n is the number of bases in the oligonucleotide.

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the T_(m) is 57° C. The T_(m) of a DNA duplex decreases by 1-1.5°C. with every 1% decrease in sequence complementarity.

Hybridisation under high stringency conditions may involve performingthe hybridisation at a temperature of Tm-15 or higher. Moderatestringency may be considered to be Tm-25 to Tm-15. Low stringency may beconsidered to be Tm-35 to Tm-25.

Accordingly, nucleotide sequences can be categorized by an ability tohybridise to a target sequence under different hybridisation and washingstringency conditions which can be selected by using the above equation.The T_(m) may be used to provide an indicator of the strength of thehybridisation.

The concept of distinguishing sequences based on the stringency of theconditions is well understood by the person skilled in the art and maybe readily applied.

Sequences exhibiting 95-100% sequence complementarity may be consideredto hybridise under very high stringency conditions, sequences exhibiting85-95% complementarity may be considered to hybridise under highstringency conditions, sequences exhibiting 70-85% complementarity maybe considered to hybridise under intermediate stringency conditions,sequences exhibiting 60-70% complementarity may be considered tohybridise under low stringency conditions and sequences exhibiting50-60%% complementarity may be considered to hybridise under very lowstringency conditions.

Nucleic Acids and Polypeptides

In this specification, a nucleic acid encoding an ING4 polypeptide maybe any nucleic acid (DNA or RNA) having a nucleotide sequence having aspecified degree of sequence identity to SEQ ID NO: 4 or SEQ ID NO: 5,to an RNA transcript of any one of these sequences, to a fragment of anyone of the preceding sequences or to the complementary sequence of anyone of these sequences or fragments. Alternatively a nucleic acidencoding an ING4 polypeptide may be one that hybridises to one of thesesequence under high or very high stringency conditions. The specifieddegree of sequence identity may be from at least 60% to 100% sequenceidentity. More preferably, the specified degree of sequence identity maybe one of at least 65%, 70%, 75%, 80%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% identity.

In this specification, an ING4 polypeptide may be any peptide,polypeptide or protein having an amino acid sequence having a specifieddegree of sequence identity to SEQ ID NO: 3 or to a fragment of one ofthese sequences. The specified degree of sequence identity may be fromat least 60% to 100% sequence identity. More preferably, the specifieddegree of sequence identity may be one of at least 65%, 70%, 75%, 80%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity.

Administration

The HSV for use in the present invention may be formulated asmedicaments and pharmaceutical compositions for clinical use and maycomprise a pharmaceutically acceptable carrier, diluent or adjuvant. Thecomposition may be formulated for topical, parenteral, systemic,intravenous, intra-arterial, intramuscular, intrathecal, intraocular,intratumoral, subcutaneous, oral or transdermal routes of administrationwhich may include injection. Injectable formulations may comprise theselected compound in a sterile or isotonic medium. Medicaments andpharmaceutical compositions may be formulated in fluid or solid (e.g.tablet) form. Fluid formulations may be formulated for administration byinjection to a selected region of the human or animal body.

Administration is preferably in a “therapeutically effective amount”,this being sufficient to show benefit to the individual. The actualamount administered, and rate and time-course of administration, willdepend on the nature and severity of the disease being treated.Prescription of treatment, e.g. decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors, andtypically takes account of the disorder to be treated, the condition ofthe individual patient, the site of delivery, the method ofadministration and other factors known to practitioners. Examples of thetechniques and protocols mentioned above can be found in Remington'sPharmaceutical Sciences, 20th Edition, 2000, pub. Lippincott, Williams &Wilkins.

Alternatively, targeting therapies may be used to deliver the activeagent more specifically to certain types of cell, by the use oftargeting systems such as antibody or cell specific ligands. Targetingmay be desirable for a variety of reasons; for example if the agent isunacceptably toxic, or if it would otherwise require too high a dosage,or if it would not otherwise be able to enter the target cells.

HSV capable of targeting cells and tissues are described in(PCT/GB2003/000603; WO 03/068809), hereby incorporated in its entiretyby reference.

A composition may be administered alone or in combination with othertreatments, either simultaneously or sequentially dependent upon thecondition to be treated.

The invention includes the combination of the aspects and preferredfeatures described except where such a combination is clearlyimpermissible or expressly avoided.

Aspects and embodiments of the present invention will now beillustrated, by way of example, with reference to the accompanyingfigures. Further aspects and embodiments will be apparent to thoseskilled in the art. All documents mentioned in this text areincorporated herein by reference.

BRIEF DESCRIPTION OF THE FIGURES

Embodiments and experiments illustrating the principles of the inventionwill now be discussed with reference to the accompanying figures inwhich:

FIG. 1.

FIG. 1 shows a 1% agarose/TAE gel showing PCR products amplified fromeither liver (lane 1) or placental (lanes 2 and 3) cDNA libraries. Thec750 bp ING4 cDNA is arrowed. Lane M=2-log DNA ladder.

FIG. 2.

FIG. 2 shows a Western blot probed with an anti-ING4 antibody at 1:500.The arrow indicates the position in lanes 1 and 2 of the 29 kDA ING4protein present in whole cell extracts from BHK cells infected with 1pfu/cell HSV1716ING4. This band is also faintly visible in cellsinfected with 0.1 pfu/cell HSV1716ING4 (lanes 3 and 4) but is absentfrom mock infected cells (lane 5).

FIG. 3.

Survival data from groups of six mice bearing CP70 subcutaneous tumourimplants following intratumoral injections on days 1 and 3 of PBS,HSV1790 or HSV1716ING4. HSV1716 expressing ING4 improves tumour kill inmice with subcutaneous ovarian tumour implants compared to HSV1716alone. Reduced expression of ING4 is associated with glioma progression.ING4 prevents new growth by inhibition of angiogenesis.

FIG. 4.

Tumour growth data from groups of six mice bearing A431 subcutaneoustumour implants following intratumoral injections on days 1 and 3 ofPBS, HSV1716 or HSV1716ING4. HSV1716 expressing ING4 reduces tumourburden in mice with subcutaneous SCC tumour implants compared to HSV1716alone.

FIG. 5.

Survival data from groups of six mice bearing A431 subcutaneous tumourimplants following intratumoral injections on days 1 and 3 of PBS,HSV1716 or HSV1716ING4. HSV1716 expressing ING4 improves tumour kill inmice with subcutaneous SCC tumour implants compared to HSV1716 alone.

FIG. 6.

Median survival. HSV1716 expressing ING4 extends survival times in micewith subcutaneous SCC tumour implants compared to HSV1716 alone.

FIG. 7.

Tumour growth data from groups of ten mice bearing A431 subcutaneoustumour implants following intravenous injections on days 1 and 3 of PBS,1×10⁶ pfu HSV1716 or 1×10⁶ pfu HSV1716ING4.

DETAILED DESCRIPTION OF THE INVENTION

Specific details of the best mode contemplated by the inventors forcarrying out the invention are set forth below, by way of example. Itwill be apparent to one skilled in the art that the present inventionmay be practiced without limitation to these specific details.

An HSV1716 Variant Expressing the Tumour Suppressor Gene Inhibitor ofNew Growth 4

Methods and Results

An HSV1716 variant expressing the ING4 tumour suppressor gene wasconstructed as follows. Primers for the PCT amplification of ING4 weredesigned using the human ING4 sequence deposited in the NCBI atncbi.nlm.nih.gov under Accession No. NM_(—)016162 GI:38201669). Theforward primer GAGAATTCGCGGCCGCGATGGCTGCGGGGATGTATTTG (SEQ ID No.1)hybridises at the ATG start codon of ING4 (in bold) and incorporatesEcoR1 and Not1 restriction sites (underlined) upstream of thetranslation start site. The reverse primerAGTCTAGACTCGAGCTATTTCTTCTTCCGTTCTTGGGA (SEQ ID No.2) hybridises 36nucleotides downstream (ING4 sequence in bold) from the TAG stop codonof ING4 and incorporates Xho1 and Xba1 sites (underlined). The c750 bpcDNA encoding ING4 (SEQ ID No.5) was successfully amplified fromcommercially available human liver and placental cDNA libraries (FIG. 1)and, as a stronger band was obtained with the placental cDNA (lanes 2and 3) library, this was used for ING4 cloning.

The PCR product was ligated into the pGEM-Teasy vector and sequencingconfirmed a 100% match with NM016162. The PCR product was then digesteddirectly with EcoR1 followed by Xba1 and ligated into the likewisedigested mammalian expression vector pCDNA4-myc-His (Invitrogen,Paisley, UK). Positive clones were identified by BamHI digestion ofminiprepped DNA (there is an internal ING4 BamHI site at nucleotide 608)and the ING4 expression cassette (CMV-IE promoter from plasmid plus ING4cDNA) was excised from a positive clone by NruI/XhoI digestion, bluntended using Klenow and cloned into the BglII digested, blunt ended andCIAP treated RL1 shuttle vector pRL1del/gfp used for the production ofHSV1716 variants by homologous recombination. RL1del/gfp is a modifiedversion of pRL1-del containing an expression cassette for gfp (PGK-gfp).RL1-del is a promoterless cloning vector, suitable for generatingICP34.5 null HSV-1. It contains a HSV-1 fragment formerly consisting ofthe RL1 gene and its flanking sequences with the majority of the RL1gene removed and replaced with a multi-cloning sequence (MCS). Thetransgene to be inserted into the RL1 loci is ligated into the MCS ofRL1-del and homologous recombination with HSV-1 DNA, driven by the RL1flanking sequences, results in concomitant deletion of the ICP34.5 openreading frame and incorporation of the appropriate transgene. To assistin plaque purification of recombinant viruses, the green fluorescentprotein gene is also inserted into the MCS of RL1-del.

RL1-del contains the HSV-1 BamHI k DNA fragment 123459-129403 whichincludes the RL1 gene and its flanking sequences cloned into the BamHIsite of plasmid pGem-3Zf (Promega, Southampton, UK). The 477 bpPflMI/BstEII fragment from the RL1 ORF (125292-125769) has been removedto inactivate the ICP34.5 gene and replaced with a MCS providing variousrestriction enzymes sites including those for BglII, NruI and XhoI.

RL1-del is described in WO2005/049844.

To create RL1-del/gfp, the 1.3 kbp blunt-ended EcoRI/AflII fragment thatcontains the PGK promoter/gfp gene was obtained by restriction digestionfollowed by Klenow treatment from the vector pSNRG (OligoEngine,Seattle, Wash., USA) and ligated into the RL1-del vector cut with therestriction enzyme NruI then alkaline phosphatase treated.

Insertion of the ING4 expression cassette in pRL1-del/gfp was confirmedby restriction enzyme digests and 50 μg of plasmid were linearized byScaI digestion and, after column clean-up using a GFX kit (GEHealthcare, Little Chalfont, UK), were used in conjunction with HSV-1DNA to cotransfect BHK cells. RL1-del/gfp/ING4 and viral DNA (c100 ug)were mixed with 20 μl lipofectamine 2000 in 250 μl DMEM/F12 serum-freemedium and added to a 60 mm plate which contained 50% confluent BHKcells. After 4 hours of incubation at 37° C. the medium was removed andthe cells shocked for exactly 4 minutes with 25% DMSO. After 3 washeswith 5 ml culture medium the cells were returned to 37° C. with 5 ml BHKmedium and left for 72 hours. Cells were then scraped into thesupernatant, the mixture sonicated in a sonicator bath for 2 minutes andstored at −70° C. until required. Serial dilutions were then plated outon Vero cells in 60 mm dishes, individual green fluorescent plaques werepicked, added to 1 ml culture medium and sonicated in a sonicator bathfor 2 minutes before serial dilutions were again plated out on Verocells. Plaque purification was repeated 6 times before stocks ofHSV1716ING4 were produced. The presence of the ING4 expression cassettesin the RL1 loci of HSV1716 was confirmed by both Southern blotting usingthe AluI/RsaI ICP34.5 fragment from plasmid pGEM34.5 and by PCR usingprimers which amplify across the ICP34.5 deleted region of HSV1716. Toconfirm expression of the inserted ING4 transgene confluent monolayersof BHK cells in 60 mm plates were infected with either 0.1 or 1 pfu/cellHSV1716ING4 and, after 24 hours, whole cells extracts were prepared bythe addition of 0.2 ml SDS PAGE sample buffer. SDS PAGE/Western blottingusing an ING4 antibody (Abcam, Cambridge, UK) identified a c29 kDaprotein in the cells infected with 1 pfu/cell HSV1716ING 4 (FIG. 2,lanes 1 and 2). A weaker band was observed in the cells infected with0.1 pfu/cell HSV1716ING4 (FIG. 2; lanes 3 and 4) and this protein wasabsent from mock infected cells (FIG. 2, lane 5).

EXAMPLE 1 In vivo Experiments

Intratumoral Injection of Nude Mice Bearing Subcutaneous Tumour Implants

In an initial experiment, 6 mice with subcutaneous implants of the CP70ovarian tumour cell line were injected intratumorally with 1×10⁷ pfuHSV1716ING4 on days 1 and 3 and tumour growth and survival was monitoreddaily. Control mice were either injected with HSV1790, an HSV1716variant expressing the enzyme nitroreductase, or were injected with asimilar volume of PBS. Survival data is shown in FIG. 3.

Of the 6 mice treated with HSV1716ING4, 1 tumour showed completeregression, 4 tumours grew at a slower rate than the untreated tumoursand 1 tumour became ulcerated and the mouse was removed from theanalysis. Using survival times (as measured by when the tumour reachedthe upper acceptable limit) to assess each of the treatments, animalstreated with the ING 4 virus had an average survival of 23.5 dayscompared to animals treated with no virus or those treated with HSV1790which had survival times of 11 or 18 days respectively. Log rankcomparison of the survival curves shown in FIG. 3 demonstratedsignificant differences with a p value of 0.03. Note that no CB1954 wasadministered to mice infected with HSV1790 and, under thesecircumstances this virus is equivalent to HSV1716.

In a subsequent experiment, groups of 6 mice bearing subcutaneousimplants of human SCC A431 tumour cells were given injections on Days 1and 3 of either 1×10⁷ pfu HSV1716ING4 or HSV1716 or an equivalent volumeof PBS and tumour growth and survival were determined daily.

FIG. 4 shows the average tumour volumes and demonstrated that the grouptreated with HSV1716ING 4 had a smaller average tumour volume thaneither the group treated with no virus or with the parental HSV1716.Statistical analysis showed that the differences in tumour volumesbetween HSV1716ING4 and HSV1716 and between HSV1716ING4 and no virus atday 15 were significantly different with P=0.03 and P=0.007respectively.

FIG. 5 shows the KM survival graph for the tumour-bearing mice treatedwith no virus, HSV1716 or HSV1716ING 4. Mice treated with no virus had amedian survival of 8 days, those treated with HSV1716 had a mediansurvival of 12 days and those treated with HSV1716ING 4 had a mediansurvival of 21 days. Comparison of the FIG. 5 curves by log rankanalysis shows that the difference between them is significant withp=0.004 thus clearly demonstrating in this tumour model that HSV1716ING4significantly reduced tumour burdens and enhanced survival.

Intravenous Injection

Groups of 10 mice with subcutaneous human SCC A431 tumours wereintravenously injected with PBS (no virus) or with 1×10⁶ pfu HSV1716 orHSV1716ING4 on days 1 and 3 and tumour growth and survival was monitoreddaily. After intravenous injection HSV1716ING4 significantly reducedtumour growth compared to no virus controls or to unmodified HSV1716(FIG. 6). On day 10 post injection, the mean tumour volume forHSV1716-injected mice was 421±55 mm² compared to 193±152 mm² forHSV1716ING4 injected mice and, using Student's t test, this differenceis highly significant with p=0.0022. Similarly, on days 14 or 20, theaverage tumour volumes for HSV1716-injected mice were 1096±402 mm² or1253±155 mm² respectively versus 476±448 mm² or 776±438 mm² respectivelyfor mice receiving HSV1716ING4 and, using Student's t test, both thesedifferences are significant with p=0.0125 or 0.0162 respectively. Micereceiving no virus or 1×10⁶ pfu HSV1716 had median survivals ofapproximately 14 days, whereas mice receiving HSV1716ING4 had a mediansurvival of 17 days with 4/10 mice surviving beyond day 20 by which timeall the PBS- or HSV1716-injected mice had been sacrificed. A number oftumours from each of the groups of mice were removed at sacrifice and,after mechanical homogenisation, virus in the tumour extract wastitrated with the results presented in Table 1.

Virus was readily extracted from all three tumours taken from the micewhich received intravenous HSV1716ING4 but was only detected in 1/3 micewhich received HSV1716 and the amounts of virus detected in theHSV1716ING4 tumours were approximately 1000-fold greater than the amountextracted from the HSV1716 tumour suggesting better replication ofHSV1716ING4 than HSV1716 within the tumour. Previous studies, albeit inimmunocompetent rats, have shown that only 0.001% of intravenouslyinjected virus lodges in the tumour 24 hours after administration(Schellingerhout et al 1998, 2000) and, assuming that this provides areasonable approximation for SCID mice, then, of the 2×10⁶ pfuadministered intravenously, only 20 pfu will reach the tumour initially.Assuming that there is no difference in biodistribution between HSV1716and HSV1716ING4, the above data demonstrates that HSV1716ING4 issignificantly better at replication within human A431 SCC tumourimplants than HSV1716 and that this enhanced propagation improvessurvival. One of the HSV1716ING4 mice survived until day 30, by whichtime the tumour had stopped growing and was almost completely regressed.After sacrifice of this mouse, all its organs were harvested includingresidual tumour tissue and, following mechanical homogenisation andtitration, no virus was found in any of these tissues suggestingcomplete recovery and viral clearance in this mouse. Indeed, there wasno evidence of viral toxicity in any of the mice which receivedHSV1716ING4 indicating that the virus retains the restricted replicationcompetence of the parental HSV1716 with propagation limited to theactively dividing tumour cells.

EXAMPLE 2 In vitro Experiments

Propagation of HSV1716ING4 on Different Cell Lines

The above in vivo data suggests that HSV1716ING4 demonstrates enhancedoncolysis by replicating with higher efficiency than unmodified HSV1716and this was confirmed in vitro using a variety of different cells linesinfected at low multiplicities of infection. Cell lines used were BHK,Vero, A431 human SCC, CP70 human ovarian tumour, MDA-MB-468 human breastadenocarcinoma, Ovcar3 human ovarian carcinoma and HuH7 humanhepatocellular carcinoma and propagation of HSV1716ING4 in these lineswas compared with wild-type HSV-1 17+, HSV1716, HSV1716ING4 andHSV1716EGFR (an EGFR-targeted HSV1716 variant that expresses a targetingmoiety approximately the same molecular size as ING4).

Cells were plated out in 60 mm dishes and after 24 hours they wereinfected with approximately 100 pfu (BHK only) or 1000 pfu (all othercell types) HSV-1 17+, HSV1716, HSV1716ING4 or HSV1716EGFR. Dilutions ofeach virus preparation for these infections were titrated on BHK cellsto confirm accurately the amounts of input virus. Each virus infectionon each cell type was performed in quadruplicate at least. After 72hours of infection, cells and medium were harvested, subjected to onefreeze/thaw cycle (−70° C.) and titrated on BHK cells. The results inthe Tables 2-8 below are reported in yields/input virion withstatistical comparisons made using ANOVA.

For BHK cells, the high yields of virus/input virion for HSV-1 17+,HSV1716 and HSV1716ING4 are very similar indicating that these threeviruses have similar replication efficiencies in this cell type and theyields, equivalent to approximately 5×10⁶ viruses per input virion, areprobably the maximum output achievable from a 100 pfu infection of BHKcells in this experiment. For HSV1716EGFR yields/input virus wereapproximately 10-fold less than for HSV-1 17+, HSV1716 and HSV1716ING4suggesting that, in this experiment, this virus replicates lessefficiently in BHK cells, see Table 2.

In Vero cells, HSV1716ING4 replicates more efficiently than HSV-1 17+,HSV1716 or HSV1716EGFR with at least a 2-fold increase in yield/inputvirion, see Table 3. Comparisons of the yield/input virion forHSV1716ING4 with each of the yields/input virion for HSV-1 17+, HSV1716or HSV1716EGFR indicated that these differences are significant. ANOVAanalysis of the data in Table 3 gives significant p values of p<0.01,p<0.05 or p<0.001 respectively for HSV1716ING4 compared to HSV-1 17+,HSV1716 or HSV1716EGFR and, since all of these values are above the 95%confidence limit, it can be concluded that the presence of the ING4expression cassette in HSV1716ING4 improves virus replication in thiscell type.

In a separate experiment, 5×10⁵ Vero cells were plated out in 60 mm dishand were allowed to attach for 6 hours before being infected induplicate at a multiplicity of infection of 1 pfu/cell with HSV-1 17+,HSV1716, HSV1716ING4 or HSV1716EGFR. After exactly 24 hours in culture,cells and medium were harvested, subjected to one freeze/thaw cycle(−70° C.) and titrated on BHK cells. For HSV1716ING4, the yields ofvirus/infected cell were approximately twice the yields ofvirus/infected cell for HSV-1 17+, HSV1716 or HSV1716EGFR. At an inputof 1 pfu/cell, HSV1716ING4 produced 140/250 virions/infected cellcompared to 50/70 for HSV-1 17+, 80/88 for HSV1716 or 50/70 forHSV1716EGFR. Thus, during 24 hours of infection in Vero cells(equivalent to one round of virus replication), HSV1716ING4 mustreplicate more efficiently than HSV-1 17+, HSV1716 or HSV1716EGFR.

For the human ovarian cancer cell line Ovcar 3 cells, HSV1716ING4replicates more efficiently than HSV-1 17+, HSV1716 or HSV1716EGFR witha 2-4-fold increase in yield/input virion, see Table 4. Comparing theyield/input virion for HSV1716ING4 with each of the yields/input virionfor HSV-1 17+, HSV1716 or HSV1716EGFR indicates that the differences aresignificant. ANOVA analysis of the data in Table 4 gives p values all ofp<0.001 for HSV1716ING4 compared to each of HSV-1 17+, HSV1716 orHSV1716EGFR and, since all of these are highly significant with greaterthan 99.9% confidence limits, HSV1716ING4 must replicate moreefficiently in this cell type.

For the human squamous cell carcinoma cell line A431, HSV1716ING4replicates more efficiently than either HSV1716 or HSV1716EGFR with a100-fold increase in yield/input virion compared to HSV1716 and 10-foldincrease compared to HSV1716EGFR, see Table 5. Statistical comparison ofthe yield/input virion for HSV1716ING4 with the yields/input virion foreither HSV1716 or HSV1716EGFR by ANOVA gives p values of p<0.05 for bothindicating that the differences are significant with the greater than95% confidence limit indicating that, compared to the parental HSV1716or the HSV1716 variant HSV1716EGFR, HSV1716ING4 must replicate moreefficiently in this cell type. The mean yield/input virion forHSV1716ING4 is greater than the mean yield/input virion for wild typeHSV-1 17+ but the difference is not significant (p>0.05). However,although HSV-1 17+ has a higher mean yield/input virion than eitherHSV1716 of HSV1716EGFR, neither of these differences is significant(both p>0.05). Similarly, although the mean yield/input virion forHSV1716EGFR is higher than the mean yield/input virion for HSV1716, thedifference is not significant (p>0.05). When the yields/input virion forHSV1716ING4 and HSV-1 17+ are compared using the less stringentStudent's t test, the differences are significant with p=0.004suggesting that HSV1716ING4 replicates more efficiently in A431 cellsthan HSV-1 17+.

In the human breast adenocarcinoma cells, HSV1716ING4 replicates moreefficiently than HSV-1 17+, HSV1716 or HSV1716EGFR with an approximately10-fold increase in yield/input virion, see Table 6. For HSV1716ING4,comparison of the yield/input virion with each of the yields/inputvirion for HSV-1 17+, HSV1716 or HSV1716EGFR indicates that thedifferences are significant. ANOVA analysis of the data in Table 6,gives significant p values, all of p<0.001, for HSV1716ING4 compared toeach of HSV-1 17+, HSV1716 or HSV1716EGFR and these are highlysignificant with greater than 99.9% confidence limits indicating thatHSV1716ING4 replicates more efficiently in this cell line.

For the human ovarian cancer cell line CP70, HSV1716ING4 replicates moreefficiently than either HSV1716 or HSV1716EGFR with a 3-fold increase inyield/input virion compared to HSV1716 or HSV1716EGFR, see Table 7.Statistical comparison of the yield/input virion for HSV1716ING4 withthe yields/input virion for either HSV1716 or HSV1716EGFR by ANOVA givesp values of p<0.01 for both indicating that the differences aresignificant with the greater than 99% confidence limit indicating that,compared to the parental HSV1716 or the HSV1716 variant HSV1716EGFR,HSV1716ING4 must replicate more efficiently in this cell type. There wasno significant difference between the yields/input virion for HSV1716and HSV1716EGFR (p>0.05). The mean yield/input virion for HSV1716ING4was not significantly different from the mean yield/input virion forwild type HSV-1 17+ (p>0.05). However, HSV-1 17+ replicated moreefficiently than either HSV1716 or HSV1716EGFR and these differenceswere significant (p<0.01) indicating that HSV1716/HSV1716EGFR areimpaired for replication in this cell type. Importantly, expression ofING4 overcomes this impairment and returns the efficiency of replicationto wild-type levels.

In the human HuH7 hepatocellular carcinoma cells, HSV1716ING4 replicatesmore efficiently than HSV-1 17+, HSV1716 or HSV1716EGFR with anapproximately 5-fold increase in yield/input virion, see Table 8. ForHSV1716ING4, comparison of the yield/input virion with each of theyields/input virion for HSV-1 17+, HSV1716 or HSV1716EGFR indicated thatthe differences are significant. ANOVA analysis of the data in Table 8gives significant p values, all of p<0.001, for HSV1716ING4 compared toeach of HSV-1 17+, HSV1716 or HSV1716EGFR and, as these are highlysignificant with greater than 99.9% confidence limits, HSV1716ING4 mustreplicate more efficiently in this cell line.

A hallmark of the attenuated HSV1716 phenotype in vitro is the inabilityof the virus to replicate in NIH 3T6 cells whereas these cells are fullypermissive for HSV-1 17+ replication. In duplicate, NIH 3T6 cells in 60mm plates were infected with 1000 pfu HSV-1 17+, HSV1716, HSV1716ING4and HSV1716EGFR. After 72 hours of infection, cells and medium wereharvested, subjected to one freeze/thaw cycle (−70° C.) and titrated onBHK cells. No virus was detected following infection of 3T6 cells withHSV1716, HSV1716ING4 or HSV1716EGFR whereas the yields from duplicateHSV-1 17+ infections of 3T6 cells were 2.0×10⁶/3.0×10⁶ pfu. Therefore,the ability of ING4 expression to enhance the replication of HSV1716 isnot achieved at the expense of its attenuated phenotype and ING4activity within the infected cell is unable to overcome the replicativerestrictions caused by deletion of the ICP34.5 gene.

Propagation on BHK Cells Engineered to Constitutively Express ING4

To generate HSV1716ING4, the ING4 cDNA was initially cloned into themammalian expression plasmid pCDNA4/myc-HisA and this vector was used tocreate BHK cell lines which constitutively express ING4. BHK cells weretransfected with 100 ug of the ING4 expression vector or with the emptypCDNA4/myc-HisA plasmid mixed with 10 ul lipofectamine 2000 (Invitrogen)in 250 ul of serum free DMEM/F12 medium. After 72 hours of transfection,cells were trypsinized and plated out with growth medium containing 1mg/ml zeocin (Invitrogen). Cells were selected with the zeocinantibiotic for 2-3 weeks after which time individual clones were clearlyvisible. Cells were trypsinized and cloned by limiting dilutions in24-well plates. Five clones of BHK/ING4 or BHK/pCDNA4 were expanded andmaintained in appropriate medium containing 0.5 mg/ml zeocin.

Each of the clones was plated out in 60 mm dishes in medium withoutzeocin and after 24 hours they were infected with approximately 10 pfuHSV-1 17+ or HSV1716. After 72 hours of infection, cells and medium wereharvested, subjected to one freeze/thaw cycle (−70° C.) and titrated onBHK cells. Dilutions of each virus prepared for the infections were alsotitrated to confirm accurately the amounts of input virus and theresults in the Tables 9 and 10 below are reported in yields/input virionwith statistical comparisons made using Student's T test.

Propagation of HSV-1 17+ on BHK cells engineered bytransfection/antibiotic selection to express constitutively ING4 isapproximately 10-fold more efficient when compared with propagation onzeocin-resistant BHK cells produced using the empty pCDNA4.myc-Hisvector. Comparison of the means using Student's t test indicates thatthis difference is highly significant with p<0.0001 indicating a greaterthan 99.99% confidence limit that ING4 expression improves HSV-1 17+replication in BHK cells.

Propagation of HSV1716 on BHK cells engineered bytransfection/antibiotic selection to express constitutively ING4 isapproximately 10-fold more efficient when compared with propagation onzeocin resistant BHK cells generated by transfection with the emptypCDNA4.myc-His vector. Comparison of the means using Student's t testdemonstrates that this difference is highly significant with p=0.02indicating a confidence limit of 98% that ING4 expression improvesHSV1716 replication in BHK cells.

EXAMPLE 3 Preliminary in vitro Experiments

The in vivo data suggested that HSV1716ING4 replicated with higherefficiency than unmodified HSV1716. This was initially confirmed invitro using a variety of different cells lines infected at lowmultiplicities of infection, as described below. These experiments werefollowed up with the experiments described in Example 2.

Cell lines used were BHK, Vero, 3T6, A431 human SCC, CP70 ovariantumour, MDA human breast carcinoma, Ovcar3 human ovarian carcinoma andUVW human glioblastoma and they were infected principally with HSV1716,HSV1716ING4 and HSV1716EGFR.

Experiment 1. Cells were plated out in 60 mm dishes and after 24 hoursthey were infected with 1, 10 or 100 pfu HSV1716, HSV1716ING4 orHSV1716EGFR. After 72 hours of infection, cells and medium wereharvested, subjected to one freeze/thaw cycle (−70° C.) and, as each ofthese viruses expresses gfp, a TCID method was used to estimate theamounts of virus in each sample. Results are presented in Tables 11-14below.

Experiment 1 clearly demonstrates that in all cell types except 3T6cells, HSV1716ING4 replicates more efficiently than HSV1716 resulting inhigher yields of virus at inputs of 1, 10 and 100 pfu. All three virusesused failed to replicate in 3T6 cells confirming their HSV1716phenotype.

Experiment 2. Cells were plated out in 60 mm dishes and after 24 hoursthey were infected in duplicate with 5 or 20 pfu HSV1716, HSV1716ING4 orHSV1716EGFR. After 72 hours of infection, cells and medium wereharvested, subjected to one freeze/thaw cycle (−70° C.) and, as each ofthese viruses expresses gfp, a TCID method was used to estimate theamounts of virus in each sample. Results are presented in Tables 15-17below.

Again, experiment 2 clearly demonstrates that in all cell typesHSV1716ING4 replicates more efficiently than HSV1716 resulting in higheryields of progeny virus at inputs of 5 or 20 pfu. The enhancement toHSV1716ING4 propagation is more pronounced at 5 pfu virus especially inOvcar3 and A431 cells in which HSV1716ING4 yields are up to 100-foldgreater than HSV1716.

Experiment 3. Each of the above cell types was seeded in a T175 flaskand once confluent, the cells were infected with HSV-1 17+, HSV1716,HSV1716ING4 or HSV1716EGFR. After 96 hours in culture, virus washarvested from both detached cells and supernatant by high speedcentrifugation and titrated on BHK cells. The dilutions used to infectthe flasks were also titred on BHK cells to quantitate accurately theamount of input virus. For each cell type, total amounts of virus/flask,% yields from input and the % yield ratios compared to the HSV1716%yield ratio are presented in Table 18.

Conclusions

An HSV1716 variant expressing the candidate tumour suppressor gene ING4shows significantly enhanced inhibition of tumour growth and prolongedsurvival times when compared with HSV1716 alone. Indeed, although notcompared directly in the above experiments, the survival times for CP70tumour-bearing mice treated with HSV1716ING4 were similar to thoseobtained following treatment with the nitroreductase-expressing variantHSV1790 in conjunction with the prodrug CB1954. The enhanced survivaltimes in the two models is unexpected and surprising given thepostulated activities of ING4 as a subunit of various complexes involvedin regulating transcription as such a mode of action in the infectedtumour cells would not be expected to have such a dramatic effect oncytotoxicity against the background of an HSV1716 lytic infection. Thus,the virus itself efficiently kills dividing tumour cells more rapidlythan any toxic effects resulting from ING4 overexpression. Possibly,ING4 expression destroys tumour cells which are infected but areresistant to virus oncolysis but this seems unlikely as the proportionof these cells in any given tumour will not be sufficient to account forthe enhanced tumour destruction seen with HSV1716ING4.

Alternatively, ING4 expressed in the infected tumour cell results in therelease of anti-angiogenesis agents such as IL-6 or IL-8 which may actlocally on surrounding uninfected cells within the tumour to suppressangiogenesis leading to enhanced tumour destruction. Thus, according tothis hypothesis, ING4 expression will stimulate release oflocally-acting messengers from the infected cell which will act uponadjacent uninfected cells resulting in suppressed expression of genespromoting angiogenesis leading to a reduction in tumour vascularizationand enhanced tumour destruction.

Moreover, mice which received intratumoral injections of PBS into eitherCP70 human ovarian cancer cell or A431 SCC implants had median survivalsof 11 or 8 days respectively. Oncolytic infection of tumour cells byHSV1716 extended median survival in both theses models to 18 or 12 daysrespectively. Further extensions to survival times to 23.5 or 21 daysoccurred with HSV1716ING4 injections indicating that the ING4 expressionsignificantly augments HSV1716 oncolytic potency. Intravenous injectionof HSV1716ING4 also reduced tumour growth and prolonged survivalcompared to intravenously injected HSV1716 and extraction and titrationof virus from the tumour implants of sacrificed mice indicated apotential mode of action for the enhance tumour cell killing byHSV1716ING4. At least 1000-fold more viruses were extracted from tumoursof mice intravenously injected with HSV1716ING4 compared to mice treatedwith unmodified HSV1716 suggesting that the expression of ING4 in theHSV1716-infected cell improves the efficiency of virus replicationresulting in augmented progeny production. Thus, during HSV1716ING4infection, the ING4 protein conditions the cell such that it provides animproved environment for HSV1716 replication. Consequentially, moreprogeny virions are produced per tumour cell infected and thisenhancement to oncolysis reduces tumour cell numbers and promotessurvival. Experiments in vitro with a panel of different cell linesconfirmed that ING4 expression improves the efficiency of HSV1716replication in a panel of different human tumour cell lines.

BEN cells are routinely used for the growth of wild type HSV-1 17+ andHSV1716 and its variants as the cell line is an excellent substrate forviral replication and high yields are always obtained. For HSV-1 17+,HSV1716 and HSV1716ING4 approximately 5×10⁸-1×10⁸ pfu were obtained byinfecting a 60 mm plate with c100 pfu virus and there was littledifference in the yields/input virion probably because this is themaximum output for BUN cells in this culture format. Vero cells can alsobe used for HSV-1 propagation although in this experiment theyields/input virion for HSV-1 17+, HSV1716, HSV1716ING4 and HSV1716EGFRwere lower than for BUN cells. At low moi, HSV1716ING4 replicated with a2-fold higher efficiency in Vero cells compared to HSV-1 17+, HSV1716 orHSV1716EGFR and statistical comparison, using the stringent ANOVA test,of HSV1716ING4 with each of these other viruses indicated that thedifferences were significant. No significant differences were detectedin yields/input virion amongst HSV-1 17+, HSV1716 and HSV1716EGFR.Further, using a much higher moi of 1 pfu/cell to infect Vero cells forone lytic cycle, HSV1716ING4 again replicated with twice the efficiencycompared to HSV-1 17+, HSV1716 or HSV1716EGFR indicating that the growthadvantage conferred by ING4 expression occurs within a single lyticcycle.

In preliminary in vitro experiments with a panel of human tumour celllines (CP70 human ovarian tumour, MDA-MB-468 human breastadenocarcinoma, Ovcar3 human ovarian carcinoma, UVW human glioblastomaand A431 SCC cells), HSV1716ING4 demonstrated enhanced replication ineach of these cell types compared to either HSV1716 or HSV1716EGFR. Insubsequent experiments using numbers of replicates that allowed forstatistically meaningful comparisons, significantly increasedyields/input virion for HSV1716ING4 compared to yields/input virion forHSV1716 or HSV1716EGFR were obtained in CP70 human ovarian tumour,MDA-MB-468 human breast adenocarcinoma, Ovcar3 human ovarian carcinoma,HuH7 human hepatocellular carcinoma and A431 SCC cells with theHSV1716ING4 output enhanced by 2-fold, 10-fold, 5-fold, 5-fold and10-100-fold respectively in these cell types. Significant differenceswere also obtained between HSV1716ING4 and HSV-1 17+ replication inMDA-MB-468 human breast adenocarcinoma, Ovcar3 human ovarian carcinomaand HuH7 human hepatocellular carcinoma cells but there was nodifference in replication efficiency between these two viruses in CP70human ovarian tumour cells. For A431 SCC cells, HSV1716ING4 output wassignificantly increased by 100-fold or 10-fold compared to HSV1716 orHSV1716EGFR respectively and, although using ANOVA, the 2-fold higherHSV1716ING4 yield/input virion was not significantly different from thatof HSV-1 17+, the difference was significant when the means werecompared using Student's t test. As with the MDA-MB-468 human breastadenocarcinoma, Ovcar3 human ovarian carcinoma and HuH7 humanhepatocellular carcinoma cells, in A431 cells there was no significantdifferences in yields/input virion for HSV-1 17+, HSV1716 orHSV1716EGFR. Thus, in 5/5 human cancer cell lines HSV1716ING4replication was significantly more efficient compared to the parentalHSV1716 or the HSV1716 variant HSV1716EGFR (expressing a similarly sizedprotein targeting moiety) and, compared to the wild-type HSV-1 17+,HSV1716ING4 replication was improved in 4/5 of these lines.

Further, when infected with either 10 pfu HSV-1 17+ or 10 pfu HSV1716,the yields/input virion were significantly enhanced 10-fold in 5different clones of BHK cells engineered by transfection/antibioticselection to constitutively express ING4 compared to BHK cells whichderived antibiotic resistance from transfection with the empty pCDNA4vector. Such BHK cells expressing ING4 constitutively may provide animproved substrate for propagation of HSV1716 and its variants,especially those with DNA inserts that restrict viral growth.

ING4 expression during HSV-1 17+/HSV1716 infection must act to improvethe efficiency of virus replication within the cell resulting in agreater output of progeny virions per cell infected. Importantly, thisenhancement to replication efficiency by ING4 did not compromise theHSV1716 phenotype as, in the animal tumour models, HSV1716ING4 wasnon-toxic and the virus failed to replicate in vitro in theHSV1716-resistant 3T6 cell line. ING4 may act directly on virus genes toimprove their expression or interact directly with a viral protein toenhance its activity or, alternatively, it may act upon cellulargenes/proteins to condition the cellular environment such that it ismore amenable to virus replication. In vivo, this improvement to virusreplication augments the oncolytic potency of HSV1716 resulting in moreviruses for better tumour cell killing. Additionally, the otherrecognised activities of ING4, such as suppression of angiogenesisleading to a reduction in tumour vascularization, may also contribute tothe enhanced oncolytic potency of HSV1716ING4.

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TABLE 1 Amounts of virus extracted from subcutaneous A431 SCC tumoursfollowing two intravenous injection of PBS or 1 × 10⁶ pfu HSV1716 ofHSV1716ING4 on days 1 and 3. Days post 1^(st) Total number of pfutreatment administration from tumour PBS control 16 0 PBS control 16 0HSV1716 16 4.1 × 10³/2.1 × 10³ HSV1716 17 0 HSV1716 17 0 HSV1716ING4 163.6 × 10⁶/4.0 × 10⁶ HSV1716ING4 18 2.7 × 10⁶ HSV1716ING4 18 4.4 × 10⁶

TABLE 2 Yields/input virion (pfu) from BHK cells infected with 100 pfuHSV-1 17+ HSV1716, HSV1716ING4 or HSV1716EGFR. repli- cate HSV-1 17+HSV1716 HSV1716ING4 HSV1716EGFR 1 9.2 × 10⁶ 3.9 × 10⁶ 4.4 × 10⁶ 5.3 ×10⁵ 2 6.9 × 10⁶ 4.4 × 10⁶ 4.0 × 10⁶ 6.7 × 10⁵ 3 7.3 × 10⁶ 3.4 × 10⁶ 3.6× 10⁶ 7.4 × 10⁵ 4 6.6 × 10⁶ 4.8 × 10⁶ 3.7 × 10⁶ 4.5 × 10⁵ 5 6.1 × 10⁶4.4 × 10⁶ 4.7 × 10⁶ 3.3 × 10⁵ 6 1.1 × 10⁷ 6.3 × 10⁶ 4.7 × 10⁶ 2.4 × 10⁵mean ± 7.85 × 10⁶ ± 4.54 × 10⁶ ± 4.19 × 10⁶ ± 4.93 × 10⁵ ± s.d. 1.88 ×10⁶ 9.87 × 10⁵ 4.83 × 10⁵ 1.92 × 10⁵

TABLE 3 Yields/input virion (pfu) from Vero cells infected with 1000 pfuHSV-1 17+ HSV1716, HSV1716ING4 or HSV1716EGFR. repli- cate HSV-1 17+HSV1716 HSV1716ING4 HSV1716EGFR 1 5.4 × 10⁵ 9.6 × 10⁵ 6.7 × 10⁵ 1.2 ×10⁵ 2 5.1 × 10⁵ 6.2 × 10⁵ 1.0 × 10⁶ 1.3 × 10⁵ 3 8.5 × 10⁵ 4.1 × 10⁵ 9.3× 10⁵ 1.6 × 10⁵ 4 2.8 × 10⁵ 8.0 × 10⁵ 1.2 × 10⁶ 1.2 × 10⁵ 5 4.1 × 10⁵5.9 × 10⁵ 1.9 × 10⁶ 1.3 × 10⁵ 6 7.1 × 10⁵ 5.3 × 10⁵ 1.3 × 10⁶ 1.4 × 10⁵mean ± 5.50 × 10⁵ ± 6.51 × 10⁵ ± 1.20 × 10⁶ ± 1.33 × 10⁵ ± s.d. 2.04 ×10⁵ 1.97 × 10⁵ 4.21 × 10⁵ 1.51 × 10⁴

TABLE 4 Yields/input virion (pfu) from human ovarian cancer Ovcar3 cellsinfected with 1000 pfu HSV-1 17+ HSV1716, HSV1716ING4 or HSV1716EGFR.repli- cate HSV-1 17+ HSV1716 HSV1716ING4 HSV1716EGFR 1 7.3 × 10⁶ 3.7 ×10⁶ 1.6 × 10⁷ 8.5 × 10⁶ 2 9.5 × 10⁶ 3.8 × 10⁶ 1.4 × 10⁷ 5.7 × 10⁶ 3 5.9× 10⁶ 3.4 × 10⁶ 1.9 × 10⁷ 7.4 × 10⁶ 4 7.8 × 10⁶ 3.3 × 10⁶ 1.4 × 10⁷ 5.4× 10⁶ mean ± 7.63 × 10⁶ ± 3.55 × 10⁶ ± 1.58 × 10⁷ ± 6.76 × 10⁶ ± s.d.1.49 × 10⁶ 2.38 × 10⁵ 2.36 × 10⁶ 1.45 × 10⁶

TABLE 5 Yields/input virion (pfu) from A431 human SCC cells infectedwith 1000 pfu HSV-1 17+ HSV1716, HSV1716ING4 or HSV1716EGFR. replicateHSV-1 17+ HSV1716 HSV1716ING4 HSV1716EGFR 1 63000 946 87000 18400 293000 1389 39000 13000 3 57000 1088 261000 10000 4 95000 1327 11300011800 mean ± 77000 ± 1188 ± 125000 ± 13300 ± 3617 s.d. 19799 207 95708

TABLE 6 Yields/input virion (pfu) from the human breast adenocarcinomaMDA-MB-468 cell line infected with 1000 pfu HSV- 1 17+ HSV1716,HSV1716ING4 or HSV1716EGFR. replicate HSV-1 17+ HSV1716 HSV1716ING4HSV1716EGFR 1 61000 74100 910000 68000 2 69000 79000 590000 66000 357000 71000 780000 65000 4 32000 66000 680000 86000 mean ± 54750 ± 72525± 740000 ± 71250 ± 9912 s.d. 15966 5456 137356

TABLE 7 Yields/input virion (pfu) from the human ovarian cancer cellline CP70 infected with 1000 pfu HSV-1 17+ HSV1716, HSV1716ING4 orHSV1716EGFR. replicate HSV-1 17+ HSV1716 HSV1716ING4 HSV1716EGFR 1 840800 1450 536 2 1350 750 1670 353 3 2700 480 1670 360 4 1091 566 1340 4085 1500 258 1920 496 mean ± 1496 ± 718 571 ± 218 1610 ± 224 430 ± 82 s.d.

TABLE 8 Yields/input virion (pfu) for the human hepatocellular carcinomacell line HuH7 infected with 1000 pfu HSV-1 17+ HSV1716, HSV1716ING4 orHSV1716EGFR. replicate HSV-1 17+ HSV1716 HSV1716ING4 HSV1716EGFR 1 964714769 62667 1760 2 12941 14215 95556 2187 3 13129 17046 86667 3413 49976 17230 71556 2933 5 8517 17969 69333 3893 mean ± 10842 ± 16246 ±77156 ± 2837 ± 871 s.d. 2075 1650 13523

TABLE 9 Yields/input virion (pfu) from 5 different clones of eitherBHK/ING4 or BHK/pCDNA4 infected with 100 pfu HSV17+ Clone BHK/ING4BHK/pCDNA4 1 4.45 × 10⁷ 5.3 × 10⁶ 2 8.96 × 10⁷ 2.6 × 10⁶ 3 8.76 × 10⁷3.8 × 10⁶ 4 8.68 × 10⁷ 2.6 × 10⁶ 5 1.13 × 10⁸ 2.0 × 10⁶ Mean ± 8.43 ×10⁷ ± 3.26 × 10⁶ ± s.d. 2.48 × 10⁷ 1.32 × 10⁶

TABLE 10 Yields/input virion (pfu) from 5 different clones of eitherBHK/ING4 or BHK/pCDNA4 infected with 100 pfu HSV1716 Clone BHK/ING4BHK/pCDNA4 1 2.6 × 10⁷ 1.8 × 10⁶ 2 2.1 × 10⁷ 1.0 × 10⁶ 3 9.2 × 10⁶ 1.3 ×10⁶ 4 7.24 × 10⁷  2.5 × 10⁶ 5 7.36 × 10⁷  2.2 × 10⁶ Mean ± 8.724 × 10⁷ ±1.009 × 10⁸ 1.766 × 10⁶ ± 6.19 × 10⁵ s.d.

TABLE 11 Total yields from different cell types following infection with1 pfu HSV1716, HSV1716EGFR or HSV1716/ING4. virus 3T6 MDA UVW A431 CP70Vero Ovcar3 BHK 1716 0 0 0 4 × 10⁴ 2 × 10⁴ 1 × 10⁵ 1 × 10⁶ 1 × 10⁷ EGFR0 0 0 5 × 10⁴ 2 × 10⁴ 2 × 10⁵ 1 × 10⁶ 4 × 10⁶ ING4 0 1 × 10³ 1 × 10³ 6 ×10⁴ 4 × 10⁴ 4 × 10⁵ 3 × 10⁷ 2 × 10⁷

TABLE 12 Total yields from different cell types following infection with10 pfu HSV1716, HSV1716EGFR or HSV1716/ING4. virus 3T6 MDA UVW A431 CP70Vero Ovcar3 BHK 1716 0 0 1 × 10⁴ 4 × 10⁵ 6 × 10⁴ 6 × 10⁵ 1 × 10⁶ 1 × 10⁷EGFR 0 0 6 × 10³ 8 × 10⁵ 4 × 10⁴ 4 × 10⁵ 6 × 10⁷ 4 × 10⁶ ING4 0 1 × 10⁴2 × 10⁴ 7.2 × 10⁵   1 × 10⁵ 2 × 10⁷ 2 × 10⁸ 4 × 10⁷

TABLE 13 Total yields from different cell types following infection with100 pfu HSV1716, HSV1716EGFR or HSV1716/ING4. virus 3T6 MDA UVW A431CP70 Vero Ovcar3 BHK 1716 0 2 × 10³ 7.2 × 10⁴   6 × 10⁵ 3 × 10⁵ 6 × 10⁶5 × 10⁷ 1 × 10⁸ EGFR 0 3 × 10⁴ 6 × 10⁴ 6 × 10⁶ 4 × 10⁵ 8 × 10⁶ 8 × 10⁷ 8× 10⁷ ING4 0 4 × 10⁴ 4 × 10⁵ 5 × 10⁶ 8 × 10⁵ 3 × 10⁷ 4 × 10⁸ 3 × 10⁸

TABLE 14 Ratios of yields (HSV1716ING4:HSV1716) in each of the differentcell types following infections at 1, 10 or 100 pfu. Cell type input BHKVero UVW MDA CP70 A431 Ovcar3  1 pfu 2:1 4:1 n.d.* n.d. 2:1 3:2 30:1  10pfu 4:1 30:1  2:1 n.d 2:1 2:1 200:1  100 pfu 3:1 5:1 6:1 20:1 3:1 10:1 10:1 *n.d. = not determinable as yield for HSV1716 was 0.

TABLE 15 Total virus yields from different cell types followingduplicate infections with 5 pfu HSV1716, HSV1716EGFR or HSV1716/ING4.Cell type virus BHK Ovcar 3 Vero A431 CP70 MDA UVW ING4   2 × 10⁸   2 ×10⁸   5 × 10⁶ 1.25 × 10⁷  2.5 × 10⁵   2 × 10⁵ 2.5 × 10⁵ ING4 1.5 × 10⁸  5 × 10⁸ 1.5 × 10⁷   1 × 10⁷ 1.8 × 10⁵   2 × 10⁵ 2.5 × 10⁵ EGFR 7.5 ×10⁷ 1.5 × 10⁷ 1.5 × 10⁶ 2.3 × 10⁶ 1.25 × 10⁵    1 × 10⁵   5 × 10³ EGFR1.3 × 10⁸ 1.8 × 10⁷ 1.5 × 10⁶   2 × 10⁶ 1.5 × 10⁵ 1.3 × 10⁵ 7.5 × 10³1716   1 × 10⁸   2 × 10⁶ 1.8 × 10⁵ 1.3 × 10⁵ 1.5 × 10⁴ 1.2 × 10⁴   1 ×10³ 1716 3.5 × 10⁸ 2.5 × 10⁶ 1.5 × 10⁵ 1.5 × 10⁵ 1.2 × 10⁴ 1.7 × 10⁴   2× 10³

TABLE 16 Total virus yields from different cell types followingduplicate infections with 20 pfu HSV1716, HSV1716EGFR or HSV1716/ING4.Cell type virus BHK Ovcar 3 Vero A431 CP70 MDA UVW ING4 6 × 10⁸   1 ×10⁹   1 × 10⁷  1.5 × 10⁷ 1.5 × 10⁵   1 × 10⁶ 5 × 10⁴ ING4 2 × 10⁸   5 ×10⁸ 2.5 × 10⁶ 1.75 × 10⁷ 1 × 10⁵ 2.5 × 10⁵   1 × 10⁴ EGFR 1 × 10⁸ 1.5 ×10⁷   1 × 10⁶   1 × 10⁶ 1 × 10⁴ 1 × 10⁵ 0 EGFR 3 × 10⁸ 1.75 × 10⁷  1.25× 10⁶    1 × 10⁶ 3 × 10⁴ 1.25 × 10⁵   0 1716 1.75 × 10⁸   1.25 × 10⁷ 1.5 × 10⁵   1 × 10⁶ 7.5 × 10⁴   2 × 10⁴ 2 × 10³ 1716 1.5 × 10⁸   2.5 ×10⁷ 1.5 × 10⁵ 1.25 × 10⁶ 1 × 10⁵ 2 × 10⁴ 5 × 10³

TABLE 17 Ratios of yields of HSV1716ING4:HSV1716 in each of thedifferent cell types following infections at 5 or 20 pfu. Cell typeinput BHK Ovcar3 Vero A431 CP70 MDA UVW 5 pfu 2:1 100:1  30:1 100:1 10:1 20:1 25:1 5 pfu 4:1 200:1  100:1  66:1 10:1 10:1 10:1 20 pfu  4:140:1 66:1 15:1  1:1 10:1 10:1 20 pfu  1:1 50:1 20:1 50:1  2:1 50:1  5:1

TABLE 18 Total amounts of virus produced/flask, % yields (output/input)and the ratio of yield/cell type compared to the HSV1716 % yield/celltype for infection of T175 flasks with HSV-1 17+, HSV1716, HSV1716EGFRor HSV1716ING4. Input Yield % Input Yield % Virus (pfu) (pfu) yield*Ratio** (pfu) (pfu) yield* Ratio** BHK UVW 17+ 1000 5.9 × 10¹⁰ 5 × 10⁷1:1 100,000 5.9 × 10⁸ 5900  8:1 1716 690 3.5 × 10¹⁰ 5 × 10⁷ 69,000 4.8 ×10⁷ 700 EGFR 1680 3.6 × 10¹⁰ 2 × 10⁷ 1:1 168,000 2.1 × 10⁵ 2   1:350ING4 175 4.0 × 10¹⁰ 2 × 10⁸ 4:1 17,500 1.4 × 10⁸ 7714 10:1 A431 Ovcar317+ 100,000 7.2 × 10⁸ 7200 2:1 100,000 4.2 × 10⁸ 4200 10:1 1716 69,0003.4 × 10⁸ 4900 69,000 2.5 × 10⁷ 360 EGFR 168,000 6.6 × 10⁸ 3900 1:1168,000 3.9 × 10⁸ 2300  7:1 ING4 17,500 3.7 × 10⁸ 21,000 5:1 17,500 1.5× 10⁸ 8571 25:1 MDA CP70 17+ 100,000   1 × 10⁶ 10 10:1  100,000 3.8 ×10⁸ 3800 36:1 1716 69,000 4.6 × 10⁴ 1 69,000 7.6 × 10⁶ 110 EGFR 168,0001.8 × 10⁵ 1 1 168,000 9.2 × 10⁶ 55  1:2 ING4 17,500 7.2 × 10⁴ 4 4:117,500 7.3 × 10⁶ 417  4:1 Vero Input Yield % Virus (pfu) (pfu) yield*Ratio** 17+ 100,000 1.1 × 10⁸ 1100 11:1 1716 69,000 6.9 × 10⁶ 100 EGFR168,000   3 × 10⁷ 177 1.7:1  ING4 17,500 2.9 × 10⁷ 1657 16:1 *amount ofoutput virus/amount input virus **ratio of % yield virus relative to %yield HSV1716

1. An herpes simplex virus (HSV), wherein the herpes simplex virusgenome comprises a nucleic acid sequence encoding an ING4 polypeptide,wherein said nucleic acid sequence comprises a nucleic acid encoding apolypeptide that has at least 95% sequence identity to SEQ ID NO: 3,wherein the HSV is oncolytic.
 2. An herpes simplex virus according toclaim 1, wherein the nucleic acid sequence comprises SEQ ID NO: 4 or SEQID NO: 5 or nucleic acid encoding the polypeptide of SEQ ID NO:
 3. 3. Anherpes simplex virus, wherein the herpes simplex virus genome comprisesa nucleic acid sequence encoding an ING4 polypeptide, wherein saidnucleic acid sequence has 95-100% complementarity to the nucleic acidsequence of SEQ ID NO: 4 or SEQ ID NO: 5, or to a nucleic acid sequenceencoding the polypeptide of SEQ ID NO.
 3. 4. An herpes simplex virusaccording to claim 1, wherein the ING4 polypeptide is a polypeptidewhose presence in a cell infected with a herpes simplex virus leads toan increase in replication efficiency of the herpes simplex virus insaid cell.
 5. A herpes simplex virus according to claim 1, wherein theING4 polypeptide is a polypeptide whose presence in a cell infected withherpes simplex virus strain 17 or strain 1716 leads to an increase inreplication efficiency of herpes simplex virus strain 17 or 1716 in saidcell.
 6. An herpes simplex virus according to claim 1 wherein saidherpes simplex virus genome further comprises a regulatory nucleotidesequence operably linked to said nucleic acid encoding ING4, whereinsaid regulatory nucleotide sequence has a role in controllingtranscription of said ING4 polypeptide.
 7. An herpes simplex virusaccording to claim 1, wherein the herpes simplex virus isnon-neurovirulent.
 8. An herpes simplex virus according to claim 1,wherein one or both of the ICP34.5 genes are modified such that theICP34.5 gene is incapable of expressing a functional ICP34.5 geneproduct.
 9. An herpes simplex virus according to claim 1, wherein allcopies of the ICP34.5 gene are modified such that the ICP34.5 gene isincapable of expressing a functional ICP34.5 gene product.
 10. An herpessimplex virus according to claim 1 which is an ICP34.5 null mutant. 11.An herpes simplex virus according to claim 1, wherein the herpes simplexvirus lacks at least one expressible ICP34.5 gene.
 12. An herpes simplexvirus according to claim 1, wherein said nucleic acid is located in atleast one RL1 locus of the herpes simplex virus genome or is located in,or overlaps, at least one of the ICP34.5 protein coding sequences of theherpes simplex virus genome.
 13. An herpes simplex virus according toclaim 3, wherein the herpes simplex virus is oncolytic.
 14. HSV1716ING4deposited with ECACC on 15 Feb. 2008 under accession number 08021501.15. A medicament, pharmaceutical composition or vaccine comprising anherpes simplex virus as claimed in claim
 1. 16. A medicament,pharmaceutical composition or vaccine as claimed in claim 15 furthercomprising a pharmaceutically acceptable carrier, adjuvant or diluent.17. A method of lysing or killing tumour cells in vitro or in vivocomprising the step of administering to the cells an herpes simplexvirus as claimed in claim
 1. 18. An herpes simplex virus according toclaim 4, wherein the cell is selected from the group consisting of Verocells, A431 human SCC cells, CP70 human ovarian tumour cells, MDA-MB-468human breast adenocarcinoma cells, Ovcar3 human ovarian carcinoma cellsand HuH7 human hepatocellular carcinoma cells.
 19. An herpes simplexvirus according to claim 1, wherein the genome comprising the nucleicacid sequence encoding an ING4 polypeptide is the genome of HSV-1 strain17, HSV-1 strain 1716, HSV-1 strain F, or HSV-2 strain HG52.